Beta-lactamyl phenylalanine, cysteine, and serine vasopressin antagonists

ABSTRACT

Substituted 2-(azetidin-2-on-1-yl)alkoxyalkylalkanoic acids and 2-(azetidin-2-on-1-yl)arylalkylalkanoic acids, and analogs and derivatives thereof are described. Methods for using the described compounds, and pharmaceutical compositions thereof, to treat disease states responsive to antagonism of one or more vasopressin receptors are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national application under 37 C.F.R. §371(b)of International Application Serial No. PCT/US2006/027703 filed Jul. 18,2006, which claims the benefit under 35 U.S.C. §119(e) of U.S.provisional patent application Ser. No. 60/700,673, filed Jul. 19, 2005,the entirety of the disclosure of which are incorporated herein byreference.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional patent application Ser. No. 60/700,673, filed Jul. 19, 2005,the entirety of the disclosure of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to 2-(azetidin-2-on-1-yl)-substitutedalkanoic acid analogs of amino acids. In particular, the inventionrelates to such alkanoic acid analogs of phenylalanine, cysteine,homocysteine, and homoserine, and analogs and derivatives thereof. Thepresent invention also relates to methods for treating mammals in needof relief from disease states associated with and responsive to theantagonism of the vasopressin V_(1a), V_(1b), and V₂ receptors.

BACKGROUND

Arginine vasopressin (AVP) is a neurohypophyseal neuropeptide producedin the hypothalamus, and is involved in many biological processes in thecirculatory system, the peripheral nervous system (PNS), and the centralnervous system (CNS). In particular, AVP acts as a neurotransmitter inthe brain. Several pharmacologically significant vasopressin receptorsubtypes, including vasopressin V_(1a), V_(1b), and V₂, have beenidentified. Such vasopressin receptors are involved in severalpsychiatric, psychological, and behavioral disease states includingdepression, anxiety, affective disorders, and stress, as well asnon-opioid mediation of tolerance for pain. Vasopressin receptors arealso involved in a number of metabolic processes including watermetabolism homeostasis, renal function, mediation of cardiovascularfunction, and regulation of temperature in mammals.

For example, AVP plays an important role in the onset of depression, oneof the most common of the serious CNS disorders. Among the potentialtargets for treating depression is thehypothalamic-pituitary-adrenal-axis (HPA axis), which is perturbed inmany depressed patients, as well as in stress-related affectivedisorders (see, Scott and Dinan, 1998; Serradiel-Le Gal et al., 2002,the disclosures of which are incorporated herein by reference).Normalization of HPA axis function appears to be a prerequisite forsustained remission of depressive symptoms when medication is used (see,Steckler, et al., 1999, the disclosures of which are incorporated hereinby reference).

One of the signs of major depression is an elevated level of cortisoland ACTH associated with dysregulation of the HPA axis (see, Owens andNemeroff, 1993; Plotsky et al. 1998, the disclosures of which areincorporated herein by reference). Corticotropin-releasing hormone (CRH)and arginine vasopressin (AVP) are the two main ACTH secretagogues, andrecent preclinical and clinical studies have shown that AVP is importantin mediating ACTH release during chronic psychological stress (see,Scott and Dinan, 1997, 1998, the disclosures of which are incorporatedherein by reference). AVP is made in neurons localized to theparaventricular nucleus of the hypothalamus, and activation of theseneurons causes the release of AVP into the portal circulation of themedian eminence. However, the cortisol response to psychological stressappears to be regulated by AVP, but not by CRH in anxious healthy humanvolunteers (see, Boudarene et al., 1999, the disclosures of which areincorporated herein by reference). Chronic psychological stressaccompanied by dysregulation of the HPA axis may contribute to theetiology of affective disorders. It has been found that many patientswith major depression show elevated levels of AVP that decline as themental illness improves (see, van Londen et al., 1997 & 2000, thedisclosures of which are incorporated herein by reference).

AVP is also transported to the anterior pituitary where it can stimulateACTH release by interacting with a V_(1b) receptor on the cell membranesof corticotrophs. For example, rats selectively bred for highanxiety-related behavior show dysregulation in this HPA axis. Treatmentwith a V_(1b) receptor antagonist can abolish CRH-stimulated ACTHsecretion, demonstrating a shift in ACTH regulation from CRH to AVP(see, Keck et al., 1999, the disclosures of which are incorporatedherein by reference). The presence of V_(1b) receptors in severalregions of the rat CNS and mouse CNS has also been demonstrated. It istherefore believed that V_(1b) antagonists that penetrate the CNS mayhave greater therapeutic potential for stress-related affectivedisorders. Currently there are no vasopressin antagonists that are ableto cross the blood brain barrier (Serradeil-Le Gal et al. 2002). Thereis also preclincial and clinical evidence that vasopressin, actingthrough a V_(1b) receptor, contributes to a subtype of major depressionassociated with chronic stress and dysregulation of the HPA axis (see,Boudarene et al., 1999; Griebel et al., 2002; Scott and Dinan, 1997,1998, the disclosures of which are incorporated herein by reference).

It has been reported that cardiovascular disease accounts for thelargest cause of hospitalizations in individuals aged 65 years andolder. It has been demonstrated that AVP contributes to thepathophysiology and progression of heart disease, including congestiveheart failure (see, Schrier & Abraham “Hormones and hemodynamics inheart failure,” N. Engl. J. Med. 341:577-585 (1999); Thibonnier“Vasopressin receptor antagonists in heart failure,” Curr. Op.Pharmacology 3:683-687 (2003); Lee et al., “Vasopressin: A new targetfor the treatment of heart failure,” Am. Heart J. 146:9-18 (2003), thedisclosures of which are incorporated herein by reference). In addition,the coordinated physiology of the renal/cardiovascular systemscontributes to normal cardiac performance and homeostasis. Thus, AVPalso plays an important role in water and electrolytic balance,regulation of blood volume, vascular smooth muscle tone, and cardiaccontractility and metabolism. Each of these are major factors affectingthe performance of the heart and its ability to meet the demands of thebody. AVP affects all of these factors, in particular through activationof V_(1a) and V₂ receptors. Vasopressin V_(1a) receptors are localizedto vascular smooth muscle and cardiomyocytes, promoting vasoconstrictionand myocardial cell protein synthesis and growth, respectively.Vasopressin V₂ receptors are localized to the collecting ducts ofnephrons in the kidney promoting free water reabsorption. Small changesin plasma osmolarity are sensed by receptors in the hypothalamus, whichregulates the neurosecretory release of AVP from the pituitary gland.With osmotic stimulation, plasma AVP levels can rise from a basal levelof 3-4 pg/ml to 9-10 pg/ml. These modest changes in AVP neurohormonelevel, in concert with the renin-angiotensin-aldosterone system,regulate the day-to-day water and electrolyte balance in healthysubjects.

However, it has been reported that the role of AVP in the cardiovascularphysiology of healthy subjects is minimal, and for those persons,supraphysiological doses of neurohormone are needed to affect bloodpressure, cardiac contractility, and coronary blood flow. In contrast,AVP plays a substantive role in patients with heart failure. Forexample, it has been observed that basal plasma levels of AVP areelevated in patients with heart failure as compared to healthy controls,particularly those that also present with hyponatremia (see, Goldsmith,“Congestive heart failure: potential role of arginine vasopressinantagonists in the therapy of heart failure,” Congest. Heart Fail.8:251-6 (2002); Schrier and Ecder, (2001), the disclosures of which areincorporated herein by reference). Further, the impaired water diuresisin congestive heart failure (CHF) patients leading to increased bloodvolume, hyponatremia, edema, and weight gain, is linked to AVP. Withheart failure, elevations in plasma AVP lead to increased peripheralvascular resistance and pulmonary capillary wedge pressure whilereducing cardiac output and stroke volume. Further, additional evidencesuggests that AVP contributes to the hypertrophic myocardiumcharacteristic of the failing heart (see, Nakamura et al., “Hypertrophicgrowth of cultured neonatal rat heart cells mediated by vasopressinV_(1a) receptor,” Eur J Pharmacol 391:39-48 (2000); Bird et al.,“Significant reduction in cardiac fibrosis and hypertrophy inspontaneously hypertensive rats (SHR) treated with a V_(1a) receptorantagonist,” (abstract) Circulation 104:186 (2001), the disclosures ofwhich are incorporated herein by reference), and cell/molecular studieshave demonstrated that it also triggers a signaling cascade thatpromotes the myocardial fibrosis typically seen with progression of thedisease.

Structural modification of vasopressin has provided a number ofvasopressin agonists (see, Sawyer, Pharmacol. Reviews, 13:255 (1961)).In addition, several potent and selective vasopressin peptideantagonists have been disclosed (see, Lazslo et al., PharmacologicalReviews, 43:73-108 (1991); Mah and Hofbauer, Drugs of the Future,12:1055-1070 (1987); Manning and Sawyer, Trends in Neuroscience, 7:8-9(1984)). Further, novel structural classes of non-peptidyl vasopressinantagonists have been disclosed (see, Yamamura et al., Science,275:572-574 (1991); Serradiel-Le Gal et al., Journal of ClinicalInvestigation, 92:224-231 (1993); Serradiel-Le Gal et al., BiochemicalPharmacology, 47(4):633-641 (1994)). Finally, the general structuralclass of substituted 2-(azetidin-2-on-1-yl)acetic acid esters and amidesare known as synthetic intermediates for the preparation of β-lactamantibiotics (see, U.S. Pat. No. 4,751,299).

SUMMARY OF THE INVENTION

It has been discovered that certain compounds within the general classof substituted 2-(azetidin-2-on-1-yl)alkanoic acids and derivativesthereof are antagonists of vasopressin receptors, including vasopressinV_(1a), V_(1b), and V₂ receptors. Described herein are2-(azetidin-2-on-1-yl)-substituted alkanoic acid analogs ofphenylalanine, cysteine, homocysteine, and homoserine, and analogs,homologs, and derivatives thereof. Also described herein arepharmaceutical compositions that include therapeutically effectiveamounts of the alkanoic acid compounds described herein for treatingdiseases and disorders that are responsive to antagonism of one or morevasopressin receptors, such as the V_(1a), V_(1b), or V₂ receptors. Inaddition, methods useful for treating diseases and disease states thatare associated with vasopressin dysfunction, and that are responsive toantagonism of a vasopressin receptor, such as the V_(1a), V_(1b), or V₂receptors, or a combination thereof, in a mammal are described. Inaddition, processes for preparing 2-(azetidin-2-on-1-yl)-substitutedalkanoic acid analogs of phenylalanine, cysteine, homocysteine, andhomoserine, and various analogs and derivatives thereof are described.

In one illustrative embodiment of the invention, compounds of formula(I) are described:

wherein:

Q is oxygen, sulfur, or oxidized sulfur, including —S(O)— and —SO₂—;

n is 1 or 2;

A is R⁵O—, monosubstituted amino, disubstituted amino, or an optionallysubstituted nitrogen-containing heterocycle attached at a nitrogen;

R¹ is hydrogen or C₁-C₆ alkyl;

R² is hydrogen, alkyl, including C₁-C₆ alkyl, alkenyl, including C₂-C₆alkenyl, such as vinyl, allyl, and the like, alkynyl, including C₂-C₆alkynyl, such as ethynyl, propynyl, and the like, alkoxy, includingC₁-C₄ alkoxy, alkylthio, including C₁-C₄ alkylthio, halo, haloalkyl,such as trifluoromethyl, trifluorochloroethyl, and the like, cyano,formyl, alkylcarbonyl, including C₁-C₃ alkylcarbonyl, alkoxycarbonyl, ora substituent selected from the group consisting of —CO₂R⁸,—CONR⁸R^(8′), and —NR⁸(COR⁹);

R³ is an amino, amido, acylamido, or ureido group, which is optionallysubstituted; or R³ is a nitrogen-containing heterocyclyl group attachedat a nitrogen atom;

R⁴ is alkyl, including C₁-C₆ alkyl, alkenyl, including C₂-C₆ alkenyl,alkynyl, including C₂-C₆ alkynyl, cycloalkyl, including C₃-C₈cycloalkyl, cycloalkenyl, including C₃-C₉ cycloalkenyl, such aslimonenyl, pinenyl, and the like, alkylcarbonyl, including C₁-C₃alkylcarbonyl, optionally substituted aryl, optionally substitutedarylalkyl, including aryl(C₁-C₄ alkyl), optionally substitutedarylhaloalkyl, optionally substituted arylalkoxyalkyl, optionallysubstituted arylalkenyl, including aryl(C₂-C₄ alkenyl), optionallysubstituted arylhaloalkenyl, or optionally substituted arylalkynyl,including aryl(C₂-C₄ alkynyl);

R⁵ is selected from hydrogen, alkyl, including C₁-C₆ alkyl, cycloalkyl,including C₃-C₈ cycloalkyl, alkoxyalkyl, including (C₁-C₄ alkoxy)-(C₁-C₄alkyl), optionally substituted arylalkyl, including aryl(C₁-C₄ alkyl),heterocyclyl, heterocyclyl(C₁-C₄ alkyl), and R⁶R⁷N—(C₂-C₄ alkyl), whereheterocyclyl is in each occurrence independently selected fromtetrahydrofuryl, morpholinyl, pyrrolidinyl, piperidinyl, piperazinyl,homopiperazinyl, or quinuclidinyl; where said morpholinyl, pyrrolidinyl,piperidinyl, piperazinyl, homopiperazinyl, or quinuclidinyl isoptionally N-substituted with C₁-C₄ alkyl or optionally substitutedaryl(C₁-C₄ alkyl);

R^(5′) is selected from the group consisting of —SR¹⁵, —S(O)R¹⁵,—SO₂R¹⁵, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, (C₁-C₄ alkoxy)-(C₁-C₄ alkyl),optionally-substituted arylalkyl, including aryl(C₁-C₄ alkyl),heterocyclyl, heterocyclyl(C₁-C₄ alkyl), and R^(6′)R^(7′)N—(C₂-C₄alkyl); where heterocyclyl is in each occurrence independently selectedfrom tetrahydrofuryl, morpholinyl, pyrrolidinyl, piperidinyl,piperazinyl, homopiperazinyl, or quinuclidinyl; where said morpholinyl,pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl, orquinuclidinyl is optionally N-substituted with C₁-C₄ alkyl or optionallysubstituted aryl(C₁-C₄ alkyl);

R⁶ is hydrogen or alkyl, including C₁-C₆ alkyl, and R⁷ is alkyl,including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈ cycloalkyl,optionally substituted aryl, or optionally substituted arylalkyl,including aryl(C₁-C₄ alkyl); or R⁶ and R⁷ are taken together with theattached nitrogen atom to form an heterocycle, such as pyrrolidinyl,piperidinyl, morpholinyl, piperazinyl, and homopiperazinyl; where saidpiperazinyl or homopiperazinyl is optionally N-substituted with R¹³;

R^(6′) is hydrogen or alkyl, including C₁-C₆ alkyl, and R^(7′) is alkyl,including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈ cycloalkyl,optionally substituted aryl, or optionally substituted arylalkyl,including aryl(C₁-C₄ alkyl); or R^(6′) and R^(7′) are taken togetherwith the attached nitrogen atom to form an heterocycle, such aspyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, andhomopiperazinyl; where said piperazinyl or homopiperazinyl is optionallyN-substituted with R^(13′);

R⁸ and R^(8′) are each independently selected in each instance fromhydrogen, alkyl, including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈cycloalkyl, optionally substituted aryl, or optionally substitutedarylalkyl, including aryl(C₁-C₄ alkyl); or R⁸ and R^(8′) are takentogether with the attached nitrogen atom to form an heterocycle, such asoptionally substituted pyrrolidinyl, piperidinyl, morpholinyl,piperazinyl, and homopiperazinyl;

R⁹ is selected from hydrogen, alkyl, including C₁-C₆ alkyl, cycloalkyl,including C₃-C₈ cycloalkyl, alkoxyalkyl, including (C₁-C₄ alkoxy)-(C₁-C₄alkyl), optionally substituted aryl, optionally substituted arylalkyl,including aryl(C₁-C₄ alkyl), optionally substituted heteroaryl,optionally substituted heteroarylalkyl, including heteroaryl(C₁-C₄alkyl), and R⁸R^(8′)N—(C₁-C₄ alkyl);

R¹³ and R^(13′) are each independently selected from hydrogen, alkyl,including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈ cycloalkyl,alkoxycarbonyl, including C₁-C₄ alkoxycarbonyl, optionally substitutedaryloxycarbonyl, optionally substituted arylalkyl, including aryl(C₁-C₄alkyl), and optionally substituted aryloyl;

R¹⁵ is selected from the group consisting of C₁-C₆ alkyl, C₃-C₈cycloalkyl, (C₁-C₄ alkoxy)-(C₁-C₄ alkyl), optionally-substitutedaryl(C₁-C₄ alkyl), Y′—, Y′—(C₁-C₄ alkyl), and R^(6′)R^(7′)N—(C₂-C₄alkyl); and

hydrates, solvates, and pharmaceutically acceptable salts thereof;

provided that when Q is oxygen, n is 2 and R^(5′) is not —SR¹⁵,—S(O)R¹⁵, or —SO₂R⁵.

In another illustrative embodiment of the invention, compounds offormula (II) are described:

wherein:

Aryl is an optionally substituted monocyclic or polycyclic aromaticgroup;

m is 1, 2, 3, or 4; and

A, R¹, R², R³, and R⁴ are as defined in formula (I); and

hydrates, solvates, and pharmaceutically acceptable salts thereof.

In another illustrative embodiment of the invention, compounds offormula (III) are described:

wherein:

Aryl is an optionally substituted monocyclic or polycyclic aromaticgroup;

Q′ is oxygen, sulfur, or —CH₂—;

n′ is 0, 1, or 2;

m′ is 0, 1, or 2; and

A, R¹, R², R³, and R⁴ are as defined in formula (I); and

hydrates, solvates, and pharmaceutically acceptable salts thereof;

provided that when Q′ is oxygen, n′ is 2; and when Q′ is sulfur, n′ is 1or 2.

In one aspect, compounds of formula (I) are described, wherein Q isoxygen, and n is 2. In another aspect, compounds of formula (I) aredescribed, wherein Q is sulfur, and n is 1 or 2. In another aspect,compounds of formula (I) are described, wherein Q is sulfur, n is 1, andR^(5′) is alkyl or optionally substituted arylalkyl. In another aspect,compounds of formula (I) are described, wherein Q is sulfur, n is 2, andR^(5′) is alkyl or optionally substituted arylalkyl.

In one aspect of the compounds of formulae (II) and (III), Aryl isoptionally substituted phenyl, including phenyl, alkylphenyl,hydroxyphenyl, alkoxyphenyl, halophenyl, cyanophenyl, and the like;optionally substituted pyridinyl, including 2-, 3-, and 4-pyridinyl,alkyl 2-, 3-, and 4-pyridinyl, halo 2-, 3-, and 4-pyridinyl, and thelike; and optionally substituted naphthyl, including 2-, and 3-naphthyl,alkylnaphthyl, hydroxynaphthyl, alkoxynaphthyl, halonaplithyl, and thelike.

It is to be understood that various aspects of the formulae describedherein may be selected in many combinations. Illustratively, for any ofthe compounds of formulae (I), (II), or (III), compounds are selectedwhere R² is hydrogen, R⁴ is an arylalkenyl, and A is either amonosubstituted amino, a disubstituted amino, or an optionallysubstituted nitrogen-containing heterocycle. In variations, compoundsare selected where R² is hydrogen or methyl, R⁴ is an arylalkyl, and Ais either a monosubstituted amino, a disubstituted amino, or anoptionally substituted nitrogen-containing heterocycle. In anotherillustrative combination for compounds of formulae (I) and (III), R² ishydrogen, R⁴ is an arylalkyl, and Q or Q′ is sulfur. In variations, A iseither a monosubstituted amino, a disubstituted amino, or an optionallysubstituted nitrogen-containing heterocycle, and n or n′ is 1. In othervariations, R¹ is hydrogen, and in still other variations, R⁴ is morespecifically optionally substituted phenylethenyl. Is to be furtherunderstood that such variations may be further combined to definesubsets of compounds selected from the invention described herein.

In another embodiment, pharmaceutical compositions are described herein,where the pharmaceutical compositions include one or more of thecompounds described herein, including but not limited to the compoundsof formulae (I), (II), or (III), and/or2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine, cysteine,homocysteine, and homoserine, and derivatives and analogs thereofdescribed herein, and combinations thereof. The2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine, cysteine,homocysteine, and homoserine and derivatives and analogs thereof includecompounds of formulae (I), (II), or (III). The pharmaceuticalcompositions described herein also include one or more pharmaceuticallyacceptable carriers, diluents, and/or excipients. In one illustrativeaspect, pharmaceutical compositions are described that exhibit oralactivity and/or oral bioavailability. In another illustrative aspect,pharmaceutical compositions are described that allow the2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine, cysteine,homocysteine, and homoserine, and derivatives and analogs thereof tocross the blood brain barrier.

In another embodiment, methods for treating disease states responsive tothe antagonism of a vasopressin V_(1a), V_(1b), and/or V₂ receptors, ina mammal in need of such treatment are described. The methods comprisethe step of administering to the mammal a pharmaceutically effectiveamount of one or more of the compounds described herein, including butnot limited to the compounds of formulae (I), (II), or (III), and/or2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine, cysteine,homocysteine, and homoserine, and derivatives and analogs thereofdescribed herein, and combinations thereof. In another embodiment, themethods comprise the step of administering to the mammal a compositioncontaining a pharmaceutically effective amount of one or more2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine, cysteine,homocysteine, and homoserine, and derivatives and analogs thereofdescribed herein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

Illustrative disease states that are responsive to the antagonism of oneor more of the vasopressin V_(1a), V_(1b), and/or V₂ receptors, andtreatable by the methods described herein, include variousstress-related mental illnesses, depression, anxiety, affectivedisorders, obsessive-compulsive disease, impulsivity, aggressivedisorders, and the like; diseases affecting water homeostasis, renalfunction, inhibition of phosphatidyl inositol turnover, temperatureregulation, and the like; diseases associated with nausea, emesis, andpain; and various cardiovascular diseases, including congestive heartfailure, disorders or conditions associated with platelet aggregation,and the like. In addition, methods for treating other disease states andconditions treatable by, for example, oxytocin receptor antagonism,tachykinin receptor antagonism, neurokimin 1 receptor antagonism,neurokinin 2 receptor antagonism, and the like are described herein,where the method includes the step of administering to a patient in needof relief from such a disease state or condition an effective amount ofone or more 2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine,cysteine, homocysteine, and homoserine, and derivatives and analogsthereof described herein, including the compounds of formulae (I), (II),or (III); or the method includes the step of administering to a patientin need of relief from such a disease state or condition a compositiondescribed herein, where the composition includes an effective amount ofone or more 2-(azetidin-2-on-1-yl)-substituted analogs of phenylalanine,cysteine, homocysteine, and homoserine, and derivatives and analogsthereof described herein, including the compounds of formulae (I), (II),or (III), and a pharmaceutically acceptable carrier, diluent, and/orexcipient.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the human V_(1b) binding affinity (Ki=0.07 nM) of Example9B through a competitive binding assay conducted in CHO cellstransfected with human V_(1a) receptor.

DETAILED DESCRIPTION

The general chemical terms used in the formulae described herein havetheir usual ordinary meanings. For example, the term “alkyl” refers to astraight-chain or optionally branched, saturated hydrocarbon, includingbut not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl,hexyl, heptyl, octyl and the like.

The term “cycloalkyl” refers to a straight-chain or optionally branched,saturated hydrocarbon, at least a portion of which forms a ring,including but not limited to cyclopropyl, cyclobutyl, cyclopentyl,methylcyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

The term “alkenyl” refers to a straight-chain or optionally branched,hydrocarbon that includes at least one double bond, including but notlimited to vinyl or ethenyl, allyl or prop enyl, isopropenyl, 2-butenyl,2-methyl-2-propenyl, butadienyl, and the like.

The term “alkynyl” refers to a straight-chain or optionally branched,hydrocarbon that includes at least one triple bond, including but notlimited to ethynyl, propynyl, 1-butynyl, hex-4-en-2-ynyl, and the like.

The term “aryl” refers to an aromatic ring or heteroaromatic ring andincludes such groups as furyl, pyrrolyl, thienyl, pyridinyl, thiazolyl,oxazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, phenyl,pyridazinyl, pyrimidinyl, pyrazinyl, thiadiazolyl, oxadiazolyl,naphthyl, indanyl, fluorenyl, quinolinyl, isoquinolinyl, benzodioxanyl,benzofuranyl, benzothienyl, and the like.

The term “optionally substituted” refers to the replacement of one ormore, illustratively from one to about three, hydrogen atoms with one ormore substitutents. Substituents include but are not limited to suchgroups as C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkylthio, hydroxy, nitro,halo, carboxy, cyano, C₁-C₄ haloalkyl, C₁-C₄ haloalkoxy, amino,carbamoyl, carboxamido, amino, alkylamino, dialkylamino,alkylalkylamino, C₁-C₄ alkylsulfonylamino, and the like.

The term “heterocycle” refers to a non-aromatic cyclic structurepossessing one or more heteroatoms, such as nitrogen, oxygen, sulfur,and the like, and includes such groups as tetrahydrofuryl, morpholinyl,pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl, quinuclidinyl,and the like.

The term “alkoxy” refers to an alkyl or cycloalkyl substituent attachedthrough an oxygen, and includes such groups as methoxy, ethoxy, propoxy,isopropoxy, butoxy, tert-butoxy and the like.

The term “acyl” includes terms such as “alkanoyl,” and “aroyl” andrefers to alkyl, alkenyl, alkyl, aryl, and the like attached through acarbonyl group. Illustratively, acyl is formyl, acetyl, propanoyl,butanoyl, pentanoyl, cyclohexanoyl, optionally substituted benzoyl, andthe like.

The term “halo” refers to fluoro, chloro, bromo, and iodo.

The term “alkanoyloxy” includes such groups as formyloxy, acetoxy,n-propionoxy, n-butyroxy, pivaloyloxy, and like lower alkanoyloxygroups.

The terms “optionally substituted C₁-C₄ alkyl,” “optionally substitutedC₃-C₈ cycloalkyl,” and “optionally substituted C₂-C₄ alkenyl” refer toalkyl, cycloalkyl, or alkenyl, respectively, optionally substituted witha substituent as described herein, including but not limited to hydroxy,protected hydroxy, alkyl, protected carboxyl, carbamoyl, benzylthio,alkylthio, and the like.

The term “(C₁-C₄ alkyl)” as used in for example “aryl(C₁-C₄ alkyl)”,“(C₁-C₄ alkoxy)-(C₁-C₄ alkyl)”, and the like, refers to a saturatedlinear or branched divalent alkyl chain of from one to four carbonshaving for example aryl, C₁-C₄ alkoxy, and the like, as a substituentand includes such groups as for example benzyl, phenethyl, phenpropyl,α-methylbenzyl, methoxymethyl, ethoxyethyl, and the like.

The term “optionally substituted phenyl” is taken to mean a phenylradical optionally substituted with one or more substituents eachindependently selected, such as C₁-C₄ alkyl, C₁-C₄ alkoxy, hydroxy,halo, nitro, trifluoromethyl, sulfonamido, cyano, carbamoyl, amino,mono(C₁-C₄ alkyl)amino, di(C₁-C₄ alkyl)amino, C₁-C₄ alkylsulfonylamino,and indol-2-yl.

The term “protected amino” refers to amine protected by a protectinggroup that may be used to protect the nitrogen, such as the nitrogen inthe β-lactam ring, during preparation or subsequent reactions. Examplesof such groups are benzyl, 4-methoxybenzyl, 4-methoxyphenyl,trialkylsilyl, for example trimethylsilyl, and the like.

The term “protected carboxy” refers to the carboxy group protected orblocked by a conventional protecting group commonly used for thetemporary blocking of the acidic carboxy. Examples of such groupsinclude lower alkyl, for example tert-butyl, halo-substituted loweralkyl, for example 2-iodoethyl and 2,2,2-trichloroethyl, benzyl andsubstituted benzyl, for example 4-methoxybenzyl and 4-nitrobenzyl,diphenylmethyl, alkenyl, for example allyl, trialkylsilyl, for exampletrimethylsilyl and tert-butyldiethylsilyl and like carboxy-protectinggroups.

It is to be understood that in the embodiments described herein, anillustrative variation of alkyl is C₁-C₆ alkyl, such as methyl, ethyl,propyl, prop-2-yl, and the like; an illustrative variation of alkenyl isC₂-C₆ alkenyl, such as vinyl, allyl, and the like; an illustrativevariation of alkynyl is C₂-C₆ alkynyl, such as ethynyl, propynyl, andthe like; an illustrative variation of alkoxy is C₁-C₄ alkoxy, such asmethoxy, pent-3-oxy, and the like; an illustrative variation ofalkylthio is C₁-C₄ alkylthio, such as ethylthio, 3-methylbuty-2-ylthio,and the like; an illustrative variation of alkylcarbonyl is C₁-C₃alkylcarbonyl, such as acetyl, propanoyl, and the like; an illustrativevariation of cycloalkyl is C₃-C₈ cycloalkyl; an illustrative variationof cycloalkenyl is C₃-C₉ cycloalkenyl, such as limonenyl, pinenyl, andthe like; an illustrative variation of optionally substituted arylalkylis optionally substituted aryl(C₁-C₄ alkyl); an illustrative variationof optionally substituted arylalkenyl is optionally substitutedaryl(C₂-C₄ alkenyl); an illustrative variation of optionally substitutedarylalkynyl is optionally substituted aryl(C₂-C₄ alkynyl); anillustrative variation of alkoxyalkyl is (C₁-C₄ alkoxy)-(C₁-C₄ alkyl);an illustrative variation of optionally substituted heteroarylalkyl isoptionally substituted heteroaryl(C₁-C₄ alkyl); and all illustrativevariation of alkoxycarbonyl is C₁-C₄ alkoxycarbonyl.

The term “antagonist”, as used herein, refers to a full or partialantagonist. While a partial antagonist of any intrinsic activity may beuseful, the partial antagonists illustratively show at least about 50%antagonist effect, or at least about 80% antagonist effect. The termalso includes compounds that are full antagonists of the vasopressinV_(1b) receptor. It is appreciated that illustrative methods describedherein require therapeutically effective amounts of vasopressin V_(1b)receptor antagonists; therefore, compounds exhibiting partial antagonismat the vasopressin V_(1b) receptor may be administered in higher dosesto exhibit sufficient antagonist activity to inhibit the effects ofvasopressin or a vasopressin agonist.

In one aspect of the compounds of formula (I), A is monosubstitutedamino, disubstituted amino, or an optionally substitutednitrogen-containing heterocycle attached at a nitrogen.

In another aspect, compounds of formula (I) are described, wherein Q isoxygen, and n is 2. In another aspect, compounds of formula (I) aredescribed, wherein Q is sulfur, and n is 1 or 2. In another aspect,compounds of formula (I) are described, wherein Q is sulfur, n is 2, andR^(5′) is alkyl or optionally substituted arylalkyl. In another aspect,compounds of formula (I) are described, wherein Q is sulfur, n is 2, andR^(5′) is alkylthio or optionally substituted arylalkylthio.

In one aspect of the compounds of formulae (II) and (III), Aryl isoptionally substituted phenyl, including phenyl, alkylphenyl,hydroxyphenyl, alkoxyphenyl, halophenyl, cyanophenyl, and the like;optionally substituted pyridinyl, including 2-, 3-, and 4-pyridinyl,alkyl 2-, 3-, and 4-pyridinyl, halo 2-, 3-, and 4-pyridinyl, and thelike; and optionally substituted naphthyl, including 2-, and 3-naphthyl,alkylnaphthyl, hydroxynaphthyl, alkoxynaphthyl, halonaphthyl, and thelike.

In another aspect, compounds of formula (II) are described, wherein Arylis optionally substituted phenyl, including phenyl, alkylphenyl,hydroxyphenyl, alkoxyphenyl, halophenyl, cyanophenyl, and the like;optionally substituted pyridinyl, including 2-, 3-, and 4-pyridinyl,alkyl 2-, 3-, and 4-pyridinyl, halo 2-, 3-, and 4-pyridinyl, and thelike; and optionally substituted naphthyl, including 2-, and 3-naphthyl,alkylnaphthyl, hydroxynaphthyl, alkoxynaphthyl, halonaplithyl, and thelike.

In another aspect, compounds of formula (II) are described, whereinR^(5′) is optionally substituted alkyl, including optionally substitutedC₁-C₆ alkyl, C₁-C₄ alkyl, and C₁-C₂ alkyl. In another aspect, compoundsof formula (II) are described, wherein R^(5′) is optionally substitutedaryl(C₁-C₄ alkyl), including phenyl(C₁-C₄ alkyl), or optionallysubstituted aryl(C₁-C₂ alkyl).

In another aspect, compounds of formula (III) are described wherein n′and m′ are each the integer 1.

In another aspect, compounds of formulae (II) and (III) are describedwherein Aryl is optionally substituted phenyl. In another aspect,compounds of formulae (II) and (III) are described wherein m and m′ areeach the integer 1.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a monosubstituted amino. In another aspect,compounds of formula (I) are described, wherein A is a disubstitutedamino. In another aspect, compounds of formula (I) are described,wherein A is an optionally substituted nitrogen-containing heterocycleattached at a nitrogen.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is an amino group of the formula R¹⁴XN—; where R¹⁴is selected from the group consisting of hydrogen, hydroxy, alkyl,including C₁-C₆ alkyl, alkoxycarbonyl, including C₁-C₄ alkoxycarbonyl,and benzyl; and where X is selected from the group consisting of alkyl,including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈ cycloalkyl,alkoxyalkyl, including (C₁-C₄ alkoxy)-(C₁-C₄ alkyl), optionallysubstituted aryl, optionally substituted arylalkyl, including optionallysubstituted aryl(C₁-C₄ alkyl), and a group Y, Y—(C₁-C₄ alkyl), R⁶R⁷N—,and R⁶R⁷N—(C₂-C₄ alkyl), where Y is an heterocycle. In one variation ofthe compounds of formulae (I), (II), and (III), R¹⁴ is hydrogen.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a heterocycle having the formula R¹⁴XN—, whereR¹⁴ and X, are taken together with the attached nitrogen atom to formthe heterocycle, such as an heterocycle selected from the groupconsisting of pyrrolidinyl, piperidinyl, piperazinyl, andhomopiperazinyl; where the heterocycle is optionally substituted withR¹⁰, R¹², R⁶R⁷N—, or R⁶R⁷N—(C₁-C₄ allyl) as defined above.

In one variation, compounds of formulae (I), (II), and (III) aredescribed wherein R¹⁴ and X are taken together with the attachednitrogen atom to form piperidinyl optionally substituted at the4-position with hydroxy, alkyl, including C₁-C₆ alkyl, cycloalkyl,including C₃-C₈ cycloalkyl, alkoxy, including C₁-C₄ alkoxy,alkoxycarbonyl, including (C₁-C₄ alkoxy)carbonyl, hydroxyalkyloxyalkyl,including (hydroxy(C₂-C₄ alkyloxy))-(C₂-C₄ alkyl), R⁶R⁷N—, R⁶R⁷N-alkyl,including R⁶R⁷N—(C₁-C₄ alkyl), diphenylmethyl, optionally substitutedaryl, optionally substituted aryl(C₁-C₄ alkyl), or piperidin-1-yl(C₁-C₄alkyl).

In another variation, compounds of formulae (I), (II), and (III) aredescribed wherein R¹⁴ and X are taken together with the attachednitrogen atom to form piperazinyl optionally substituted at the4-position with alkyl, including C₁-C₆ alkyl, cycloalkyl, includingC₃-C₈ cycloalkyl, optionally substituted aryl, optionally substitutedarylalkyl, including optionally substituted aryl(C₁-C₄ alkyl),α-methylbenzyl, and the like, N-alkyl acetamid-2-yl, including N—(C₁-C₅alkyl)acetamid-2-yl, N-(cycloalkyl)acetamid-2-yl, including N—(C₃-C₈cycloalkyl)acetamid-2-yl, R⁶R⁷N—, R^(6′)R^(7′)N—, or alkoxycarbonyl,including (C₁-C₄ alkoxy)carbonyl.

In another variation, compounds of formulae (I), (II), and (III) aredescribed wherein A is a disubstituted amino having the formula R¹⁴XN—,where R¹⁴ and X are taken together with the attached nitrogen atom toform piperidinyl optionally substituted in the 4-position with alkyl,including C₁-C₄ alkyl, or heterocyclyl(C₁-C₄ alkyl).

In another variation, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a disubstituted amino having the formula R¹⁴XN—where R¹⁴ and X are taken together with the attached nitrogen atom toform piperidinyl optionally substituted in the 4-position withpiperadinyl(C₁-C₄ alkyl), piperazinyl(C₁-C₄ alkyl), orpyrrolidinyl(C₁-C₄ alkyl).

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a monosubstituted amino. In another aspect,compounds of formulae (I), (II), and (III) are described, wherein A is adisubstituted amino. In another aspect, compounds of formulae (I), (II),and (III)) are described, wherein A is an optionally substitutednitrogen-containing heterocycle attached at a nitrogen.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a monosubstituted amino having the formula XNH—,where X is selected from the group consisting of alkyl, including C₁-C₆alkyl, cycloalkyl, including C₃-C₈ cycloalkyl, alkoxyalkyl, including(C₁-C₄ alkoxy)-(C₁-C₄ alkyl), optionally substituted aryl, optionallysubstituted arylalkyl, including optionally substituted aryl(C₁-C₄alkyl), and a group Y, Y—(C₁-C₄ alkyl), R⁶R⁷N—, and R⁶R⁷N—(C₂-C₄ alkyl),where Y is an heterocycle.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is a disubstituted amino having the formula R¹⁴XN—;where R¹⁴ is selected from the group consisting of hydroxy, alkyl,including C₁-C₆ alkyl, alkoxycarbonyl, including C₁-C₄ alkoxycarbonyl,and benzyl; and where X is selected from the group consisting of alkyl,including C₁-C₆ alkyl, cycloalkyl, including C₃-C₈ cycloalkyl,alkoxyalkyl, including (C₁-C₄ alkoxy)-(C₁-C₄ alkyl), optionallysubstituted aryl, optionally substituted arylalkyl, including optionallysubstituted aryl(C₁-C₄ alkyl), and a group Y, Y—(C₁-C₄ alkyl), R⁶R⁷N—,and R⁶R⁷N—(C₂-C₄ alkyl), where Y is an heterocycle.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed, wherein A is an optionally substituted heterocycle having theformula R¹⁴XN—, where R¹⁴ and X are taken together with the attachednitrogen atom to form the heterocycle, such as an heterocycle selectedfrom the group consisting of pyrrolidinyl, piperidinyl, piperazinyl, andhomopiperazinyl; where the heterocycle is optionally substituted withR¹⁰, R¹², R⁶R⁷N—, or R⁶R⁷N—(C₁-C₄ alkyl) as defined above.

In another aspect, compounds of formulae (I), (II), and (III) aredescribed wherein R¹⁴ and X, are taken together with the attachednitrogen atom to form piperidinyl optionally substituted at the4-position with hydroxy, alkyl, including C₁-C₆ alkyl, cycloalkyl,including C₃-C₈ cycloalkyl, alkoxy, including C₁-C₄ alkoxy,alkoxycarbonyl, including (C₁-C₄ alkoxy)carbonyl, hydroxyalkyloxyalkyl,including (hydroxy(C₂-C₄ alkyloxy))—(C₂-C₄ alkyl), R⁶R⁷N—, R⁶R⁷N-alkyl,including R⁶R⁷N—(C₁-C₄ alkyl), diphenylmethyl, optionally substitutedaryl, optionally substituted aryl(C₁-C₄ alkyl), or piperidin-1-yl(C₁-C₄alkyl).

In another aspect, compounds of formulae (I), (II), and (III) aredescribed wherein R¹⁴ and X are taken together with the attachednitrogen atom to form piperazinyl optionally substituted at the4-position with alkyl, including C₁-C₆ alkyl, cycloalkyl, includingC₃-C₈ cycloalkyl, optionally substituted aryl, optionally substitutedarylalkyl, including optionally substituted aryl(C₁-C₄ alkyl),α-methylbenzyl, and the like, N-allyl acetamid-2-yl, including N—(C₁-C₅alkyl)acetamid-2-yl, N-(cycloalkyl)acetamid-2-yl, including N—(C₃-C₈cycloalkyl)acetamid-2-yl, R⁶R⁷N—, R^(6′)R^(7′)N—, or alkoxycarbonyl,including (C₁-C₄ alkoxy)carbonyl.

Illustrative compounds of formulae (I), (II), and (III) are describedwherein A is a disubstituted amino having the formula R¹⁴XN—, where R¹⁴and X are taken together with the attached nitrogen atom to formpiperadinyl optionally substituted in the 4-position with alkyl,including C₁-C₄ alkyl, or heterocyclyl(C₁-C₄ alkyl).

Illustrative compounds of formulae (I), (II), and (III) are described,wherein A is a disubstituted amino having the formula R¹⁴XN— where R¹⁴and X are taken together with the attached nitrogen atom to formpiperadinyl optionally substituted in the 4-position withpiperadinyl(C₁-C₄ alkyl), piperazinyl(C₁-C₄ alkyl), orpyrrolidinyl(C₁-C₄ alkyl).

Illustrative compounds of formulae (I), (II), and (III) are described,wherein R¹⁴ and X are taken together with the attached nitrogen atom toform homopiperazinyl optionally substituted in the 4-position withalkyl, including C₁-C₄ alkyl, aryl, or aryl(C₁-C₄ alkyl).

Illustrative compounds of formulae (I), (II), and (III) are described,wherein A is a disubstituted amino having the formula R¹⁴XN—, where R¹⁴and X are taken together with the attached nitrogen atom to form anheterocycle selected from the group consisting of pyrrolidinonyl,piperidinonyl, 2-pyrrolidin-1-ylmethyl)pyrrolidin-1-yl, and1,2,3,4-tetrahydroisoquinolin-2-yl.

In another aspect of the compounds of formulae (I), (II), or (III), R³is a structure selected from the group consisting of

wherein R¹⁰ and R¹¹ are each independently selected from hydrogen,optionally substituted alkyl, including C₁-C₆ alkyl, optionallysubstituted cycloalkyl, including C₃-C₈ cycloalkyl, alkoxyalkyl,including C₁-C₄ alkoxycarbonyl, alkylcarbonyloxy, including C₁-C₅alkylcarbonyloxy, optionally substituted aryl, optionally substitutedarylalkyl, including aryl(C₁-C₄ alkyl), optionally substitutedarylalkyloxy, including aryl(C₁-C₄ alkyloxy), optionally substitutedarylalkylcarbonyloxy, including aryl(C₁-C₄ alkylcarbonyloxy),diphenylmethoxy, and triphenylmethoxy; and

R¹² is selected from hydrogen, alkyl, including C₁-C₆ alkyl, cycloalkyl,including C₃-C₈ cycloalkyl, alkoxycarbonyl, including C₁-C₄alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionallysubstituted arylalkyl, including aryl(C₁-C₄ alkyl), and optionallysubstituted aryloyl.

In another aspect, compounds of formulae (I), (II), or (III) aredescribed, wherein R³ is a structure selected from the group consistingof

wherein R¹⁰, R¹¹, and R¹² are as defined herein.

In another aspect, compounds of formulae (I), (II), or (III) aredescribed, wherein R³ is a structure selected from the group consistingof

where R¹⁰, R¹¹, and R¹² are as defined herein.

In another aspect, compounds of formulae (I), (II), or (III) aredescribed, wherein R³ is a structure selected from the group consistingof

where R¹⁰, R¹¹, and R¹² are as defined herein.

It is to be understood that the foregoing embodiments, aspects, andvariations of the invention described herein may be combined in allpossible ways to define additional embodiments, aspects, and variations.For example, in another aspect, formulae (I), (II), or (III) aredescribed wherein A is a disubstituted amino having the formula R¹⁴XN—,where R¹⁴ and X are taken together with the attached nitrogen atom toform piperidinyl optionally substituted in the 4-position with alkyl,including C₁-C₄ alkyl, or heterocyclyl(C₁-C₄ allyl); and R³ is thestructure

wherein R¹⁰ and R¹¹ are as defined herein.

The compounds described herein possess an azetidinone core structurethat includes asymmetric carbon atoms at C(3) and C(4), creating fourstereoisomeric configurations, as illustrated by the following:

The compounds described herein may therefore exist as singlediastereomers, as a racemic mixture, or as a mixture of variousdiastereomers. It is understood that in some applications, certainstereoisomers or mixtures of stereoisomers may be used, while in othersapplications, other stereoisomers or mixtures of stereoisomers may beused. In some embodiments, a single stereoisomer is described, such asthe azetidinone core structure having the (3S,4R)-diastereomericconfiguration.

It is also understood that the α-carbon bearing R¹ is also chiral.Furthermore, the groups selected for R¹, R², R³, R⁴, and A may alsoinclude chiral centers. For example, when R³ is 4-substitutedoxazolidin-2-on-3-yl, the 4-position of that ring is asymmetric. Inaddition, when R³ is 2,5-disubstituted oxazolidin-4-on-3-yl or1,2,5-trisubstituted imidazolidin-4-on-3-yl, the 2- and 5-carbons ofthose rings are each asymmetric. Finally, when R³ is succinimido and oneof R¹⁴ and R¹⁵ is hydrogen, the carbon bearing the non-hydrogensubstituent is also asymmetric. Therefore, additional stereoisomers arecollectively represented by formulae (I), (II), or (III). Whilecompounds possessing all combinations of stereochemical purity arecontemplated by the present description, it is appreciated that in manycases at least one of these chiral centers described above may bepresent as a single absolute configuration in a compound describedherein. In one illustrative aspect, the compounds described herein havethe (αR,3S,4R) absolute configuration or the (αS,3S,4R) absoluteconfiguration.

Illustrative embodiments of the compounds described herein includeclasses of compounds of formulae (I), (II), or (III) where:

A is R⁵O—;

A is R⁵O—, and R⁵ is C₁-C₆ alkyl;

A is R⁵O—, and R⁵ is optionally substituted aryl(C₁-C₄ alkyl);

A is a monosubstituted amino of the formula XNH—;

A is a disubstituted amino having the formula R¹⁴XN—;

A is XNH— or R¹⁴XN, and X is optionally substituted aryl(C₁-C₄ alkyl);

A is XNH— or R¹⁴XN, and X is R⁶R⁷N—(C₁-C₄ alkyl);

A is XNH— or R¹⁴XN, X is R⁶R⁷N—(C₁-C₄ alkyl), and R⁶ and R⁷ are takentogether with the attached nitrogen atom to form an heterocycle;

A is R¹⁴XN, and R¹⁴ and X are taken together with the attached nitrogenatom to form an heterocycle;

A is R¹⁴XN, R¹⁴ and X are taken together with the attached nitrogen atomto form an heterocycle, and the heterocycle is optionally substitutedwith an optionally substituted heterocyclyl(C₁-C₄ alkyl);

A is R¹⁴XN, R¹⁴ and X are taken together with the attached nitrogen atomto form a piperadinyl, and the piperadinyl is optionally substituted inthe 4-position with heterocyclyl(C₁-C₄ alkyl), includingpiperadinyl(C₁-C₄ alkyl), piperazinyl(C₁-C₄ alkyl), andpyrrolidinyl(C₁-C₄ alkyl);

A is XNH— or R¹⁴XN—, and X is optionally substituted aryl(C₁-C₄ alkyl);

A is XNH— or R¹⁴XN—, X is optionally substituted aryl(C₁-C₄ alkyl), andaryl is optionally substituted phenyl;

R¹ is hydrogen;

R¹ is C₁-C₆ alkyl;

R¹ is C₁-C₂ alkyl;

R² is hydrogen;

R² is C₁-C₂ alkyl;

R² is methyl;

R² is methylthio;

R² is cyano;

R³ is 4-substituted oxazolidin-2-on-3-yl;

R³ is 4,5-disubstituted oxazolidin-2-on-3-yl;

R³ is 2-substituted oxazolidin-4-on-3-yl;

R³ is 2-substituted imidazolidin-4-on-3-yl;

R³ is 1,2-disubstituted imidazolidin-4-on-3-yl;

R³ is 5-substituted imidazolidin-2-on-1-yl;

R³ is 4,5-disubstituted imidazolidin-4-on-1-yl;

R⁴ is optionally substituted 2-aryleth-1-yl;

R⁴ is optionally substituted 2-arylethen-1-yl;

R^(5′) is C₁-C₆ alkyl;

R^(5′) is optionally substituted aryl(C₁-C₄ alkyl);

Further illustrative embodiments of the compounds described hereininclude classes of compounds of formula (II) where A, R⁵, X, R¹⁴, R¹,R², R³, and R⁴ are as described above; and where Aryl is phenyl,substituted phenyl, or 4-substituted phenyl.

It is appreciated that the classes of compounds described above may becombined to form additional illustrative classes. Further combinationsof the classes of compounds described above are contemplated in thepresent invention.

Further illustrative classes of compounds are described by the followingformula:

wherein Ar is optionally-substituted phenyl, optionally-substitutedpyridinyl, optionally-substituted furyl, or optionally-substitutedthienyl; A is nitrogen-containing heterocycle attached at the nitrogenatom, which is optionally substituted with heterocyclyl(C₁-C₄ alkyl);and R^(5′) is optionally substituted arylalkyl, including aryl(C₁-C₄alkyl).

Further illustrative classes of compounds are described by the followingformulae

wherein Ar is optionally-substituted phenyl, optionally-substitutedpyridinyl, optionally-substituted furyl, or optionally-substitutedthienyl; A is nitrogen-containing heterocycle attached at the nitrogenatom, which is optionally substituted with heterocyclyl(C₁-C₄ alkyl);and R^(5′) is optionally substituted arylalkyl, including aryl(C₁-C₄alkyl).

Further illustrative classes of compounds are described by compounds ofthe following formula

wherein Ar is optionally-substituted phenyl, optionally-substitutedpyridinyl, optionally-substituted furyl, or optionally-substitutedthienyl; A is nitrogen-containing heterocycle attached at the nitrogenatom, which is optionally substituted with heterocyclyl(C₁-C₄ alkyl); nis 1, 2, or 3; and Aryl is optionally substituted phenyl or optionallysubstituted naphthyl.

In another embodiment, the compounds described herein include a basicamino group. Such amines are capable of forming salts with a variety ofinorganic and organic acids to form pharmaceutically acceptable acidaddition salts. It is appreciated that in cases where compounds of theformulae described herein are oils rather than solids, those compoundscapable of forming addition salts that are solid will ease the handlingand administration of the compounds described herein. Acids commonlyemployed to form such salts are inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,and the like, and organic acids, such as p-toluenesulfonic acid,methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, acetic acid, and thelike. Examples of such pharmaceutically acceptable salts thus are thesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,β-hydroxybutyrate, glycollate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and the like. Preferred pharmaceutically acceptable salts arethose formed with hydrochloric acid, trifluoroacetic acid, maleic acidor fumaric acid.

The compounds described herein are useful in methods for antagonism ofthe vasopressin V_(1a), V_(1b), and V₂ receptors. Such antagonism isuseful in treating a variety of disorders and diseases that have beenlinked to this receptor in mammals. Illustratively, the mammal to betreated by the administration of compounds described herein is human.

In another embodiment, compounds are also described herein that crossthe blood brain barrier. It is appreciated that compounds that cross theblood brain barrier may have wider application in treating variousdisease states that are responsive to vasopressin antagonism. Forexample, it is to be understood that there are currently recognizeddistinct subtypes within depressive illness.

In another embodiment, processes for preparing compounds of formulae(I), (II), or (III) are described. In one aspect, processes forpreparing compounds of the formulae:

are described, wherein W is QR^(5′) or Aryl as described in variousembodiments herein; Ar¹ is optionally substituted aryl, or optionallysubstituted heteroaryl; and R¹, R², R⁴, n, and A, are as described invarious embodiments herein. The processes include the step of reacting acompound of the formula:

with a compound of the formula:

wherein W′ is -QR^(5′) or Aryl as described in various embodimentsherein, or W′ is a protected form of QR^(5′) or Aryl that may bedeprotected or converted into -QR^(5′) or Aryl. In one aspect of theprocess, when Q is oxygen, n is 2. In one variation, processes forpreparing compounds of the above formula, wherein R⁴ is optionallysubstituted arylethenyl are described. The processes include the step ofreacting a compound of the formula (A) with a compound of the formula:

wherein W′ is -QR^(5′) or Aryl as described in various embodimentsherein, or W′ is a protected form of QR⁵ or Aryl that may be deprotectedor converted into -QR^(5′) or Aryl. In one aspect of the process, when Qis oxygen, n is 2.

Generally, the 2-(azetidinon-1-yl)acetic acid esters and amides, and theanalogs and derivatives thereof described herein may be prepared bysyntheses known in the art, as well as by the various methods describedherein. As illustrated for compounds of formulae (I), (II), and (III),the 2-(azetidinon-1-yl)alkanedioic acid esters described herein areobtainable by the 2+2 cycloaddition of an appropriately substitutedacetic acid derivative thereof (i), and an imine ester (ii) upontreatment with a base in an appropriately selected solvent, as describedin Synthetic Scheme I, where Z is hydroxyl or a leaving group, and theinteger n, and the moieties A, R¹, R², R³, and R⁴ are as previouslydescribed. The term “leaving group” as used hereinafter refers to asubstitutent, such as halo, acyloxy, benzoyloxy and the like, present onan activated carbon atom that may be replaced by a nucleophile. Thechemistry described in Synthetic Scheme I is applicable to imines (ii)bearing ester, thioester, or amide moieties.

The preparation of the appropriate imines (ii), preparation ofrepresentative examples of the required acetyl halides or anhydrides(i), and the cycloaddition procedure are generally described in U.S.Pat. Nos. 4,665,171 and 4,751,299, the disclosures of which are herebyincorporated by reference. It is appreciated that when Q is sulfur incompounds (ii-a), or an oxidized form thereof, such as sulfoxide orsulfone, certain reaction conditions may not be compatible. In thosecases, appropriately selected protecting groups may be used to blockunintended reactions of the sulfur. Illustrative sulfur protectinggroups are described in Greene & Wuts “Protective Groups in OrganicSynthesis,” 2d Ed., John Wiley & Sons, New York, 1991, the disclosure ofwhich is incorporated herein by reference.

In one illustrative variation, R³ is a 4-substitutedoxazolidin-2-on-3-yl or 1,4,5-trisubstituted imidazolidin-2-on-3-yl.Those compounds of formulae (I), (II), and (III) requiring W to be a4-substituted oxazolidin-2-on-3-yl or 1,4,5-trisubstitutedimidazolidin-2-on-3-yl are prepared from the corresponding(4-substituted oxazolidin-2-on-3-yl) or (1,4,5-trisubstitutedimidazolidin-2-on-3-yl)acetyl halide or anhydride. The acid halide oranhydride is available from an appropriately substituted glycine. Theglycine is first converted to the carbamate and then reduced to providethe corresponding alcohol. The alcohol is then cyclized to the4-substituted oxazolidin-2-one, which is subsequently N-alkylated with ahaloacetic acid ester. The ester is hydrolyzed, and the resulting acidis converted to the acetyl halide or anhydride (i). Illustrative of theoxazolidinones that are included in this synthetic route, and subsequentsynthetic routes described herein, include the following commerciallyavailable compounds.

R¹⁰ R¹¹ (4R)-methyl (5S)-phenyl (4R)-methyl diphenyl (4S)-phenyl(5R)-phenyl (4S)-phenyl diphenyl (4S)-benzyl dimethyl (4S)-tert-butyldiphenyl (4R)-benzyl H (4R)-isopropyl H (4S)-methyl (5R)-phenyl(4R)-phenyl (5S)-phenyl (4S)-tert-butyl H (4S)-1H-indol-3-ylmethyl H(4S)-benzyl H (4S)-diphenylmethyl H (4S)-isopropyl H

Illustrative of the imidazolidinones and imidazolidindiones that areincluded in this synthetic route, and subsequent synthetic routesdescribed herein, include the following commercially availablecompounds.

R¹⁰ R¹¹ R¹² H H 2-methoxyphenyl H H 4-methoxyphenyl H H 2-methylphenyl HH 3-methylphenyl H H 4-methylphenyl H H acetyl H H phenyl (4S)-phenyl(5R)-methyl methyl H H methyl H H tert-butyl

R¹⁰ R¹¹ R¹² (2S)-tert-butyl (5S)-benzyl (5S)-benzyl dimethyl methyl H(2R)-tert-butyl methyl

R¹⁰ R¹² Q methyl phenyl S

In another illustrative variation, R³ is 2,5-disubstitutedoxazolidin-4-on-3-yl or 1,2,5-trisubstituted imidazolidin-4-on-3-yl.Those compounds of formulae (I), (II), and (III) requiring R³ to be2,5-disubstituted oxazolidin-4-on-3-yl or 1,2,5-trisubstitutedimidazolidin-4-on-3-yl are prepared from the corresponding(2,5-disubstituted oxazolidin-4-on-3-yl) or (1,2,5-trisubstitutedimidazolidin-4-on-3-yl)acetyl chlorides or anhydrides respectively.Reaction conditions useful for preparing these reagents are described inU.S. Pat. No. 4,772,694, hereby incorporated by reference. Briefly, therequired oxazolidinone or imidazolidinone is obtained from anα-hydroxyacid or an α-aminoacid, respectively. The imidazolones areprepared by converting the α-aminoacid, (R¹¹)—CH(NH₂)CO₂H, to anamino-protected amide and then condensing the amide with an aldehyde,(R¹⁰)—CHO, in the presence of an acid to form the 3-protectedimidazolidin-4-one, where R¹⁰ and R¹¹ are as defined above. The1-position may be functionalized with an appropriate reagent tointroduce R¹² and the 3-position deprotected, where R¹² is as definedabove. The imidazolidin-4-one ring is then alkylated with a haloaceticacid ester, the ester diesterified, and the resulting acetic acidconverted to the desired acid halide or anhydride (i). The requiredoxazolidinones are prepared in an analogous manner from thecorresponding α-hydroxyacid, (R¹¹)—CH(OH)CO₂H.

In another illustrative variation, R³ is succinimido. Those compounds offormulae (I), (II), and (III) requiring R³ to be succinimido areprepared from the corresponding 2-(succinimido)acetyl halide oranhydride. The chemistry to prepare these reagents is described in U.S.Pat. No. 4,734,498, hereby incorporated by reference. Briefly, thesereagents are obtained from tartaric acid or, when one of R¹⁰ and R¹¹ ishydrogen, from malic acid. Tartaric acid is acylated or O-alkylated, thecorresponding diacyl or di-O-alkyl tartaric acid is treated with an acidanhydride to form the succinic anhydride, and reaction of this succinicanhydride with an ester of glycine to form first the noncyclic halfamide ester which is then cyclized to the 3,4-disubstitutedsuccinimidoacetic acid ester. The ester group is diesterified and theresulting acid converted to the corresponding acid halide or anhydride(i). The mono-substituted succinimidoacetyl halide or anhydride isobtained with malic acid via succinic anhydride formation followed bysuccinimide formation as described above.

In another illustrative variation, R³ is an N-substituted amine or anN′-substituted urea. Those compounds of formulae (I), (II), and (III)requiring R³ to be an N-substituted amine or an N′-substituted urea maybe prepared from the corresponding phthalimido protected 3-aminoanalogs. The phthalimide protecting group may be removed usingconventional procedures, such as by treatment with hydrazine, and thelike. Once liberated, the amine may be alkylated with any one of avariety of alkyl and cycloalkyl halides and sulfates, such as methyliodide, isopropylbromide, diethyl sulfate, cyclopropylmethylbromide,cyclopentyliodide, and the like. Such amines may also be acylated withacid halides, acid anhydrides, isocyanates, isothiocyanates, such asacetyl chloride, propionic anhydride, methylisocyanate,3-trifluoromethylphenylisothiocyanate, and the like.

The bases to be used in Synthetic Scheme I include, among others,aliphatic tertiary amines, such as trimethylamine and triethylamine,cyclic tertiary amines, such as N-methylpiperidine andN-methylmorpholine, aromatic amines, such as pyridine and lutidine, andother organic bases such as 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU).

The solvents useful for reactions described in Synthetic Scheme Iinclude, among others, dioxane, tetrahydrofuran, diethyl ether, ethylacetate, dichloromethane, chloroform, carbon tetrachloride, benzene,toluene, acetonitrile, di-methyl sulfoxide and N,N-dimethylformamide. Itis appreciated that any desired stereochemical configuration of thesecompounds may be prepared using the processes described herein, byselecting the desired configuration at each chiral center noted above.Such a selection may be accomplished by using optically pure startingmaterials, or by separating mixtures of optical isomers at convenienttimes during the syntheses of the two foregoing formulae using standardtechniques.

The azetidinone ring may also be prepared with a deficit of substituentsR², R³, R⁴, or the R¹-substituted N-alkanedioic acid or alkoxyalkanoicacid moiety, but possessing substituents capable of being elaboratedthrough subsequent chemical transformation to such groups described forcompounds of formulae (I), (II), and (III). In general, azetidinones maybe prepared via N—C(4) cyclization, such as the cyclization ofacylhydroxamates (iv) to azetidinone intermediates (v), as depicted inScheme II, and illustrated for compounds of formula (I), where R¹, R²,R³, R⁴, and A are as defined above, according to the procedure ofMattingly et al. in J. Am. Chem. Soc. (1979), 101, 3983 and Accts. Chem.Res. (1986), 19, 49, the disclosures of which are incorporated herein byreference. It is appreciated that other hydroxamates, such asalkylhydroxamates, aryl hydroxamates, and the like, are suitable forcarrying out the cyclization.

Subsequent chemical transformation of the acyloxyazetidinone (v) tointroduce for example an R¹-substituted amino acid imine usingconventional procedures will illustratively provide compounds offormulae (I), (II), and (III).

An alternative cyclization to form intermediate azetidinones, which maybe further elaborated to compounds of formulae (I), (II), and (III) mayoccur by oxidative cyclization of acylhydroxamates (vi) to intermediateazetidinones (vii), as illustrated in Synthetic Scheme III, andillustrated for compounds of formula (I), where R² and R³ are as definedabove and L is a leaving group such as halide, according to theprocedure of Rajendra and Miller in J. Org. Chem. (1987), 52, 4471 andTetrahedron Lett. (1985), 26, 5385, the disclosures of which areincorporated herein by reference. The group R in Scheme III representsan alkyl or aryl moiety selected to provide R⁴, as defined above, uponsubsequent transformation. For example, R may be the group ArCH₂— whereAr is an optionally substituted aryl group, as in (vii-a), such thatoxidative elimination of HBr will provide the desired R⁴, such as astyryl group, as in (vii-b). It is appreciated that elaboration of R toR⁴ is not necessarily performed immediately subsequent to thecyclization and may be performed conveniently after other steps in thesynthesis of compounds of formulae (I), (II), and (III). It is furtherappreciated that alternatives to the acylhydroxamates shown, such asalkylhydroxamates, aryl hydroxamates, and the like, are suitable forcarrying out the cyclization.

Still other useful intermediates, such as the azetidinonyl acetic acidderivatives (x), may be converted into compounds of formulae (I), (II),and (III), as illustrated for the synthesis of compounds of formula (I)in Synthetic Scheme IV, and illustrated for compounds of formula (I),where R¹, R², R³, R⁴, A, and n are as defined above. Introduction of theR¹ moiety, and a carboxylic acid derivative R^(5′)-Q-(CH₂)_(n)— forcompounds of formula (I), may be accomplished by alkylation of the anionof (x).

Acetic acid derivative (x) is deprotonated and subsequently alkylatedwith an alkyl halide corresponding to R¹—Z, where Z is a leaving group,to provide intermediate (xi-a). Illustratively, the anion of (xi-a) maybe alkylated with a compound Z′—(CH₂)_(n)QR^(5′), where Z′ is a leavinggroup, to provide compounds of formula (I).

A solution of the 2-(3,4-disubstituted azetidin-2-on-1-yl)acetic acidderivative (x) or (xi) in an appropriate solvent, such astetrahydrofuran, dioxane, or diethyl ether, is treated with anon-nucleophilic base to generate the anion of (x) or (xi),respectively. Suitable bases for this transformation include lithiumdiisopropylamide, lithium 2,2,6,6-tetramethylpiperidinamide, or lithiumbis(trimethylsilyl)amide. The anion is then reacted with an appropriateelectrophile to provide the desired compounds. Illustrativeelectrophiles represented by the formula Aryl-(CH₂)_(n)—Z provide thecorresponding compounds.

The foregoing synthetic procedures may be used generally for thepreparation of the compounds described herein, including but not limitedto the serine, homoserine, cysteine, homocysteine, phenylalanine,homophenylalanine, and further homologs thereof. In addition, those samesynthesis may be used to prepare analogs and derivatives of thereof,such as tyrosine analogs, naphthyl and substituted naphthyl analogs,oxidized embodiments of the sulfur containing compounds, disulfideembodiments of the sulfur containing compounds, oxidized disulfideembodiments of the sulfur containing compounds, and the like.

Alternatively, disulfide embodiments may be prepared from serine andhomoserine compounds by converting the terminal hydroxyl group into aleaving group, such as a halo, alkyl or arylsulfonyl, acyloxy, and thelike to prepare the compounds of formula (I) or (III), as shown inScheme V and illustrated for compounds of formula (I).

Serine and homoserine compounds may converted into compounds of formula(xii), where L is a leaving group using conventional processes.Compounds (xii) may then be converted in compounds (xiii) by treatingwith a sulfide anion, disulfide anion, sulfoxide anion, or sulfonylanions, wherein R¹⁵ is as defined herein, and m is 1 or 2. It isappreciated that other nucleophiles, including sulfonylthio may also beused to displace the leaving group L in the preparation of compounds(xiii).

Alternatively, oxidized sulfur atoms may be synthesized by nucleophilicdisplacement treatment of the thioether or disulfide compounds describedherein by treating with an oxidizing agent, such as a peroxy-basedoxidizing agent, and the like. Typical oxidizing agents include hydrogenperoxide, peroxides, peroxy acids, and the like. In the case ofdisulfide oxidation, it is appreciated that only one of the two sulfuratoms may undergo oxidation. It is further appreciated that under suchcircumstances, the sulfur atom adjacent to the more electron-donatinggroup may be selectively oxidized.

Alternatively, oxidized sulfur atoms may be synthesized by conventionaltreatment of the thioether or disulfide compounds described herein bytreating with an oxidizing agent, such as a peroxy-based oxidizingagent, and the like. Typical oxidizing agents include hydrogen peroxide,peroxides, peroxy acids, and the like. In the case of disulfideoxidation, it is appreciated that only one of the two sulfur atoms mayundergo oxidation. It is further appreciated that under suchcircumstances, the sulfur atom adjacent to the more electron-donatinggroup may be selectively oxidized.

The compounds prepared as described in Synthetic Schemes I-V may be purediastereomers, mixtures of diastereomers, or racemates. The actualstereochemical composition of the compound will be dictated by thespecific reaction conditions, combination of substituents, andstereochemistry or optical activity of the reactants employed. It isappreciated that diasteromeric mixtures may be separated bychromatography or fractional crystallization to provide singlediastereomers if desired, using standard methods. Particularly, thereactions described in Synthetic Schemes II, III, and IV create a newchiral center at the carbon bearing R¹.

Alternative syntheses have also been described, including the synthesesof several members of the structural class of substituted2-(azetidin-2-on-1-yl)acetic acid esters and amides for the preparationof β-lactam, antibiotics. See, e.g., U.S. Pat. No. 4,751,299.

The following preparations and examples further illustrate the compoundsthat are illustrative of the invention described herein, including thesynthesis of such compounds, but such exemplary preparations andexamples and are not intended to and should not be interpreted to limitthe scope of the invention in any way. Unless otherwise indicated, allreactions were performed at ambient temperature, and all evaporationswere performed in vacuo. All of the compounds described below werecharacterized by standard analytical techniques, including nuclearmagnetic resonance spectroscopy (NMR) and mass spectral analysis (MS).

EXAMPLES

Each of the Examples prepared below exhibited an ¹H NMR spectrumconsistent with the assigned structure. Mass spectral analysis was alsoperformed using FAB⁺ to observe the corresponding (M+H)⁺ parent ion.

Example 1A

(4(S)-phenyloxazolidin-2-on-3-yl)acetyl chloride. A solution of 1.0equivalent of (4(S)-phenyloxazolidin-2-on-3-yl)acetic acid (Evans, U.S.Pat. No. 4,665,171) and 1.3 equivalent of oxalyl chloride in 200 mLdichloromethane was treated with a catalytic amount of anhydrousdimethylformamide (85 μL/milliequivalent of acetic acid derivative)resulting in vigorous gas evolution. After 45 minutes all gas evolutionhad ceased and the reaction mixture was concentrated under reducedpressure to provide the title compound as an off-white solid afterdrying for 2 h under vacuum.

Example 1B

(4(R)-phenyloxazolidin-2-on-3-yl)acetyl chloride. Prepared following theprocedure of Example 1A, except that(4(R)-phenyloxazolidin-2-on-3-yl)acetic acid was used instead of(4(S)-phenyloxazolidin-2-on-3-yl)acetic acid (see, Evans & Sjogren,Tetrahedron Lett. 26:3783 (1985)).

Example 1C

2-(4(S)-Phenyloxazolidin-2-on-3-yl)propanoyl chloride. A solution of 1equivalent of Example 3A and 1.3 equivalent of oxalyl chloride in 200 mLCH₂Cl₂ (150 mL/g of propanoic acid derivative) was treated with acatalytic amount of anhydrous DMF (85 μL/mmole of propanoic acidderivative) resulting in vigorous gas evolution. After 45 min., all gasevolution had ceased and the reaction mixture was concentrated underreduced pressure to provide the title compound as an off-white solidafter drying for 2 h. under vacuum.

Example 2A

Methyl (4(S)-phenyloxazolidin-2-on-3-yl)acetate. A solution of(4(S)-phenyloxazolidin-2-on-3-yl)acetic acid (1 g, 4.52 mmol) (Evans inU.S. Pat. No. 4,665,171) in 20 mL of anhydrous methanol was treatedhourly with 5 equivalents of acetyl chloride, for a total of 20equivalents. The resulting solution was stirred overnight. The residueobtained after evaporation of the MeOH was redissolved in 30 mL ofCH₂Cl₂ and treated with 50 mL of saturated aqueous Na₂CO₃. The organiclayer was evaporated and dried (MgSO₄) to yield the title compound as acolorless oil (1.001 g, 94%); ¹H NMR (CDCl₃) δ 3.37 (d, J=18.0 Hz, 1H),3.69 (s, 3H), 4.13 (t, J=8.3 Hz, 1H), 4.28 (d, J=18.0 Hz, 1H), 4.69 (t,J=8.8 Hz, 1H), 5.04 (t, J=8.4 Hz, 1H), 7.26-7.29 (m, 2H), 7.36-7.42 (an,3H).

Example 2B

Methyl 2-(4(S)-phenyloxazolidin-2-on-3-yl)propanoate. A solution ofExample 2A (1 g, 4.25 mmol) in 10 mL of anhydrous THF at −78° C. wastreated with 4.68 mL (4.68 mmol) of a 1 M solution of lithiumbis(trimethylsilyl)amide in THF. The reaction mixture was stirred for 1h. at about −70° C. before adding MeI (1.59 mL, 25.51 mmol). Uponcomplete conversion of the azetidinone, the reaction was quenched withsaturated aqueous NH₄Cl and partitioned between EtOAc and water. Theorganic layer was washed sequentially with saturated aqueous sodiumbisulfite, and saturated aqueous NaCl. The resulting organic layer wasdried (MgSO₄) and evaporated to afford the title compound (a mixture ofdiasteromers) as a white solid (1.06 g, 93%); ¹H NMR (CDCl₃) δ 1.07/1.53(d/d, J=7.5 Hz, 3H), 3.59/3.74 (s/s, 3H), 3.85/4.48 (q/q, J=7.5 Hz, 1H),4.10-4.14 (m, 1H), 4.60-4.64/4.65-4.69 (m/m, 1H), 4.88-4.92/4.98-5.02(m/m, 1H), 7.24-7.40 (m, 5H).

Example 3A

2-(4(S)-Phenyloxazolidin-2-on-3-yl)propanoic acid. To a solution ofExample 2B (1 g, 4.01 mmol) in 35 mL of MeOH was added, at 0° C., 14.3mL (12.04 mmol) of a 0.84 M solution of LiOH in water. The reactionmixture was then stirred for 3 h. at ambient temperature. Upon completehydrolysis of the azetidinone, the MeOH was removed by evaporation, thecrude residue dissolved in CH₂Cl₂ and treated with saturated aqueousNaCl. The resulting organic layer was dried (MgSO₄) and evaporated toafford the title compound (racemic mixture) as a white solid (0.906 g,96%); ¹H NMR (CDCl₃) δ 1.13/1.57 (d/d, J=7.5 Hz, 3H), 3.75/4.50 (q/q,J=7.5 Hz, 1H), 4.10-4.16 (m, 1H), 4.62-4.72 (m, 1H), 4.92-5.03 (m, 1H),7.32-7.43 (m, 5H).

Example 4

General procedure for amide formation from an activated esterderivative. N-Benzyloxycarbonyl-L-aspartic acid β-t-butyl esterα-(3-trifluoromethyl)benzylamide. A solution ofN-benzyloxycarbonyl-L-aspartic acid β-t-butyl esterα-N-hydroxysuccinimide ester (1.95 g, 4.64 mmol, Advanced ChemTech) in20 mL of dry tetrahydrofuran was treated with 0.68 mL (4.74 mmol) of3-(trifluoromethyl)benzyl amine. Upon completion (TLC, 60:40hexanes/ethyl acetate), the mixture was evaporated, and the resultingoil was partitioned between dichloromethane and a saturated aqueoussolution of sodium bicarbonate. The organic layer was evaporated to give2.23 g (quantitative yield) of the title compound as a white solid; ¹HNMR (CDCl₃) δ 1.39 (s, 9H), 2.61 (dd, J=6.5 Hz, J=17.2 Hz, 1H), 2.98(dd, J=3.7 Hz, J=17.0 Hz, 1H), 4.41 (dd, J=5.9 Hz, J=15.3 Hz, 1H),4.50-4.57 (m, 2H), 5.15 (s, 2H), 5.96-5.99 (m, 1H), 6.95 (s, 1H),7.29-7.34 (m, 5H), 7.39-7.43 (m, 2H), 7.48-7.52 (m, 2H).

Example 5

General procedure for hydrolysis of a tert-butyl ester. A solution oftert-butyl ester derivative in formic acid, typically 1 g in 10 mL, isstirred at ambient temperature until no more ester is detected by thinlayer chromatography (dichloromethane 95%/methanol 5%), a typicalreaction time being around 3 hours. The formic acid is evaporated underreduced pressure; the resulting solid residue is partitioned betweendichloromethane and saturated aqueous sodium bicarbonate. The organiclayer is evaporated to give an off-white solid that may be used directlyfor further reactions, or recrystallized from an appropriate solventsystem if desired.

Example 6

General procedure for amide formation from a carboxylic acid.Illustrated for N-Benzyloxycarbonyl-D-aspartic acid β-t-butyl esterα-(3-trifluoromethyl)benzylamide. A solution of 1 g (2.93 mmol) ofN-benzyloxycarbonyl-D-aspartic acid β-t-butyl ester monohydrate(Novabiochem) in 3-4 mL of dichloromethane was treated by sequentialaddition of 0.46 mL (3.21 mmol) of 3-(trifluoromethyl)benzylamine, 0.44g (3.23 mmol) of 1-hydroxy-7-benzotriazole, and 0.62 g (3.23 mmol) of1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. After atleast 12 hours at ambient temperature or until complete as determined bythin layer chromatography (95:5 dichloromethane/methanol eluent), thereaction mixture was washed sequentially with a saturated aqueous sodiumbicarbonate solution and with distilled water. The organic layer wasevaporated to give 1.41 g (quantitative yield) of the title compound asan off-white solid; ¹H NMR (CDCl₃) δ 1.39 (s, 9H); 2.61 (dd, J=6.5 Hz,J=17.2 Hz, 1H); 2.98 (dd, J=4.2 Hz, J=17.2 Hz, 1H); 4.41 (dd, J=5.9 Hz,J=15.3 Hz, 1H); 4.50-4.57 (m, 2H); 5.10 (s, 2H); 5.96-6.01 (m, 1H);6.91-7.00 (m, 1H); 7.30-7.36 (m, 5H); 7.39-7.43 (m, 2H); 7.48-7.52 (m,2H).

Example 6A

N-tButyloxycarbonyl-(S)-(benzyl)-D-cysteine-[4-(2-(1-piperidyl)ethyl)]piperidinenamide.N-t-Butyloxycarbonyl-(S)-Benzyl-D-cysteine (0.289 g, 0.93 mmole) and4-[2-(1-piperidyl)ethyl]piperidine (0.192 g, 0.98 mmole) were combinedin dichloromethane (20 mL) according to the procedure of Example 6 togive 0.454 g (quantitative yield) as an off-white solid. ¹H NMR (CDCl₃)δ 0.89-1.15 (m, 2H); 1.39-1.44 (m, 16H); 1.54-1.61 (m, 4H); 1.62-1.71(m, 1H); 2.21-2.35 (m, 5H); 2.49-2.58 (m, 2H); 2.66-2.74 (m, 1H);2.79-2.97 (m, 1H); 3.67-3.76 (m, 3H); 4.48-4.51 (m, 1H); 4.72-4.75 (m,1H); 5.41-5.44 (m, 1H); 7.19-7.34 (m, 5H).

Example 7A

N-[(9H-Fluoren-9-yl)methoxycarbonyl]-O-(benzyl)-D-serine t-Butyl ester.N-[(9H-Fluoren-9-yl)methoxycarbonyl]-O-(benzyl)-D-serine (0.710 g, 1.70mmole) in dichloromethane (8 mL) was treated with t-butyl acetate (3 mL)and concentrated sulfuric acid (40 μL) in a sealed flask at 0° C. Uponcompletion (TLC), the reaction was quenched with of dichloromethane (10mL) and saturated aqueous potassium bicarbonate (15 mL). The organiclayer was washed with distilled water, and evaporated. The resultingresidue was purified by flash column chromatography (98:2dichloromethane/methanol) to yield 0.292 g (77%) as a colorless oil; ¹HNMR (CDCl₃) δ 1.44 (s, 9H); 3.68 (dd, J=2.9 Hz, J=9.3 Hz, 1H); 3.87 (dd,J=2.9 Hz, J=9.3 Hz, 1H); 4.22 (t, J=7.1 Hz, 1H); 4.30-4.60 (m, 5H);5.64-5.67 (m, 1H); 7.25-7.39 (m, 9H); 7.58-7.61 (m, 2H); 7.73-7.76 (m,2H).

Example 8A

O-(Benzyl)-D-serine t-Butyl ester. Example 7A (0.620 g, 1.31 mmol) indichloromethane (5 mL) was treated with tris(2-aminoethyl)amine (2.75mL) for 5 h. The resulting mixture was washed twice with a phosphatebuffer (pH=5.5), once with saturated aqueous potassium bicarbonate, andevaporated to give 0.329 g (quantitative yield) of the title compound asan off-white solid; ¹H NMR (CD₃OD) δ 1.44 (s, 9H); 3.48 (dd, J=J′=4.2Hz, 1H); 3.61 (dd, J=4.0 Hz, J=9.2 Hz, 1H); 3.72 (dd, J=4.6 Hz, J=9.2Hz, 1H); 4.47 (d, J=12.0 Hz, 1H); 4.55 (d, J=12.0 Hz, 1H); 7.26-7.33 (m,5H).

Example 9 General Procedure for Formation of a 2-Azetidinone from anImine and an Acetyl Chloride

Step 1: General procedure for formation of an imine from an amino acidderivative. A solution of 1 equivalent of an α-amino acid ester or amidein dichloromethane is treated sequentially with 1 equivalent of anappropriate aldehyde, and a desiccating agent, such as magnesium sulfateor silica gel, in the amount of about 2 grams of desiccating agent pergram of starting α-amino acid ester or amide. The reaction is stirred atambient temperature until all of the reactants are consumed as measuredby thin layer chromatography. The reactions are typically completewithin an hour. The reaction mixture is then filtered, the filter cakeis washed with dichloromethane, and the filtrate concentrated underreduced pressure to provide the desired imine that is used as is in thesubsequent step.

Step 2: General procedure for the 2+2 cycloaddition of an imine and anacetyl chloride. A dichloromethane solution of the imine (10 mLdichloromethane/1 gram imine) is cooled to 0° C. To this cooled solutionis added 1.5 equivalents of an appropriate amine, typicallytriethylamine, followed by the dropwise addition of a dichloromethanesolution of 1.1 equivalents of an appropriate acetyl chloride, such asthat described in Example 1A (10 mL dichloromethane/1 gm appropriateacetyl chloride). The reaction mixture is allowed to warm to ambienttemperature over 1 h and is then quenched by the addition of a saturatedaqueous solution of ammonium chloride. The resulting mixture ispartitioned between water and dichloromethane. The layers are separatedand the organic layer is washed successively with 1N hydrochloric acid,saturated aqueous sodium bicarbonate, and saturated aqueous sodiumchloride. The organic layer is dried over magnesium sulfate andconcentrated under reduced pressure. The residue may be used directlyfor further reactions, or purified by chromatography or bycrystallization from an appropriate solvent system if desired.

Example 9A

tert-Butyl(2R)-(Benzyloxymethyl)-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetate.The imine prepared from 0.329 g (1.31 mmol) of O-(benzyl)-D-serinet-butyl ester (Example 8A) and cinnamaldehyde was combined with2-(4(S)-phenyloxazolidin-2-on-3-yl)acetyl chloride (Example 1A)according to the procedure of Example 9 to give 0.543 g (73%) afterflash column chromatography purification (90:10 hexanes/ethyl acetate);¹H NMR (CDCl₃) δ 1.39 (s, 9H); 3.56 (dd, J=2.7 Hz, J=9.5 Hz, 1H); 3.82(dd, J=4.8 Hz, J=9.5 Hz, 1H); 4.11 (t, J=8.3 Hz, 1H); 4.21-4.29 (m, 2H);4.50-4.58 (m, 3H); 4.71-4.78 (m, 2H); 6.19 (dd, J=9.1 Hz, J=16.0 Hz,1H); 6.49 (d, J=16.0 Hz, 1H); 7.07-7.11 (m, 1H); 7.19-7.40 (m, 14H).

Example 9B

(2S)-(Benzylthiomethyl)-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]aceticacid N-[4-[2-(piperid-1-yl)ethyl]piperidin-1-yl]amide. The imineprepared from(S)-(benzyl)-D-cysteine-[4-(2-(1-piperidyl)ethyl)]piperidinenamide,dihydrochloride (Example 1A, 0.417 g, 0.90 mmole) and cinnamaldehyde, inthe presence on triethylamine (0.26 mL, 1.87 mmole), was combined with2-(4(S)-phenyloxazolidin-2-on-3-yl)acetyl chloride (Example 1A)according to the procedure of Example 9 to give 0.484 g (76%) as anoff-white solid after recrytallization from dichloromethane/hexanes. ¹HNMR (CDCl₃) δ 0.89-1.06 (m, 2H); 1.40-1.44 (m, 5H); 1.57-1.67 (m, 6H);2.25-2.43 (m, 6H); 2.45-2.59 (m, 2H); 2.71-2.88 (m, 2H); 3.55-3.70 (m,3H); 4.11-4.17 (m, 1H); 4.37-4.47 (m, 2H); 4.54-4.61 (m, 1H); 4.64-4.69(m, 1H); 4.76-4.84 (m, 2H); 6.05-6.19 (m, 1H); 6.66-6.71 (m, 1H);7.12-7.40 (m, 15H).

Examples 9C-9AD

Shown in the following Table, may also be prepared using the proceduresdescribed herein by replacing the serine or cysteine derivativedescribed above with the one corresponding to the compounds shown below.

Exam- ple A n′ Q′ m′ 9C (3-trifluorobenzyl)amino 2 —O— 1 9D4-(3-trifluoromethylphenyl)piperazin-1-yl 2 —O— 2 9E4-(3-trifluoromethylphenyl)piperazin-1-yl 2 —O— 1 9F4-cyclohexylpiperazin-1-yl 2 —O— 2 9G4-(piperidin-1-ylmethyl)piperidin-1-yl 2 —O— 1 9H4-(piperidin-1-yl)piperidin-1-yl 2 —O— 2 9I4-[2-(piperidin-1-yl)ethyl]piperidin-1-yl 2 —O— 1 9J(3-trifluorobenzyl)amino 1 —S— 2 9K4-(3-trifluoromethylphenyl)piperazin-1-yl 1 —S— 1 9L4-(3-trifluoromethylphenyl)piperazin-1-yl 1 —S— 2 9M4-cyclohexylpiperazin-1-yl 1 —S— 1 9N4-(piperidin-1-ylmethyl)piperidin-1-yl 1 —S— 2 9O4-(piperidin-1-yl)piperidin-1-yl 1 —S— 1 9P4-[2-(piperidin-1-yl)ethyl]piperidin-1-yl 1 —S— 2 9Q(3-trifluorobenzyl)amino 2 —S— 2 9R4-(3-trifluoromethylphenyl)piperazin-1-yl 2 —S— 1 9S4-(3-trifluorometbylphenyl)piperazin-1-yl 2 —S— 2 9T4-cyclohexylpiperazin-1-yl 2 —S— 1 9U4-(piperidin-1-ylmethyl)piperidin-1-yl 2 —S— 2 9V4-(piperidin-1-yl)piperidin-1-yl 2 —S— 1 9W4-[2-(piperidin-1-yl)ethyl]piperidin-1-yl 2 —S— 2 9X(3-trifluorobenzyl)amino 0 —CH₂— 1 9Y4-(3-trifluoromethylphenyl)piperazin-1-yl 0 —CH₂— 2 9Z4-(3-trifluoromethylphenyl)piperazin-1-yl 0 —CH₂— 1 9AA4-cyclohexylpiperazin-1-yl 0 —CH₂— 2 9AB4-(piperidin-1-ylmethyl)piperidin-1-yl 0 —CH₂— 1 9AC4-(piperidin-1-yl)piperidin-1-yl 0 —CH₂— 2 9AD4-[2-(piperidin-1-yl)ethyl]piperidin-1-yl 0 —CH₂— 1

Example 10A

(2R)-(Benzyloxymethyl)-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]aceticacid. Example 9A (0.16 g, 0.28 mmol) was hydrolyzed according to theprocedure used in Example 5 to give 0.144 g (quantitative yield) as anoff-white solid; ¹H NMR (CDCl₃) δ 3.65 (dd, J=4.0 Hz, J=9.5 Hz, 1H);3.82 (dd, J=5.5 Hz, J=9.5 Hz, 1H); 4.11 (dd, J=7.8 Hz, J=8.8 Hz, 1H);4.33 (s, 2H); 4.50 (d, J=5.0 Hz, 1H); 4.57 (t, J=9.0 Hz, 1H); 4.67 (dd,J=4.0 Hz, J=5.0 Hz, 1H); 4.69 (dd, J=5.0 Hz, J=9.5 Hz, 1H); 4.75 (t,J=8.0 Hz, 1H); 6.17 (dd, J=9.3 Hz, J=15.8 Hz, 1H); 6.55 (d, J=16.0 Hz,1H); 7.09-7.12 (m, 2H); 7.19-7.42 (m, 13H).

The compound of Example 10A is used to prepare other amide and esterderivatives, such as the amides and esters represented by the group A incompounds of formulae (I), (II), and (III).

Example 11A

(S)-(benzyl)-D-cysteine-[4-(2-(1-piperidyl)ethyl)]piperidinenamide,dihydrochloride.N-tButyloxycarbonyl-(S)-(benzyl)-D-cysteine-[4-(2-(1-piperidyl)ethyl)]piperidinenamide(0.453 g, 0.93 mmole) was reacted overnight with acetyl chloride (0.78mL, 13.80 mmole) in anhydrous methanol (15 mL). The title compound wasobtained as an off-white solid by evaporating the reaction mixture todryness (0.417 g, 97%). ¹H NMR (CD₃OD) δ 0.94-1.29 (m, 2H); 1.49-1.57(m, 1H); 1.62-1.95 (m, 10H); 2.65-2.80 (m, 2H); 2.81-2.97 (m, 4H);3.01-3.14 (m, 2H); 3.50-3.60 (m, 3H); 3.81-3.92 (m, 2H); 4.41-4.47 (m,2H); 7.25-7.44 (m, 5H).

Example 12A

tert-Butyl[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetate.The imine prepared from 4.53 g (34.5 mmol) glycine tert-butyl ester andcinnamaldehyde was combined with2-(4(S)-phenyloxazolidin-2-on-3-yl)acetyl chloride (Example 1A)according to the procedure of Example 9, to give 5.5 g (30%) of Example15 as colorless crystals (recrystallized, n-chlorobutane); mp 194-195°C.

Example 13

General procedure for alklylation and/or acylation of an(azetidin-2-on-1-yl)acetate. A solution of (azetidin-2-on-1-yl)acetatein tetrahydrofuran (0.22 M in azetidinone), such as Example 12A, iscooled to −78° C. and is with lithium bis(trimethylsilyl)amide (2.2equivalents). The resulting anion is treated with an appropriate alkylor acyl halide (1.1 equivalents). Upon complete conversion of theazetidinone, the reaction is quenched with saturated aqueous ammoniumchloride and partitioned between ethyl acetate and water. The organicphase is washed sequentially with 1N hydrochloric acid, saturatedaqueous sodium bicarbonate, and saturated aqueous sodium chloride. Theresulting organic layer is dried (magnesium sulfate) and evaporated. Theresidue is purified by silica gel chromatography with an appropriateeluent, such as 3:2 hexane/ethyl acetate.

This procedure is used to prepare compounds of formulae (I), (II), and(III) by an alternate synthetic route from a common intermediate such astert-Butyl[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetate,and related compounds. This procedure is also used to prepare alkylatedand acylated analogs of the compounds described herein, such ascompounds of formulae (I), (II), and (III) wherein R¹ is other thanhydrogen. It is further appreciated that this procedure may be modifiedto introduce additional groups onto the azetidinone ring to preparecompounds described herein where R² is other than hydrogen.

It is appreciated that the epimers of these compounds at the carbonalpha to the azetidinone may also be prepared by the proceduresdescribed above, by selecting the appropriate starting materials.Further, all other compounds falling within the scope of the compoundsof formula (I), (II), and (III) may also be generally prepared by theforegoing examples.

In another embodiment, the compounds described herein are useful forantagonism of the vasopressin V_(1a), V_(1b), and V₂ receptors inmethods for treating patients suffering from disease states andconditions that are responsive to antagonism of the vasopressin V_(1a),V_(1b), and V₂ receptors. Illustratively, the methods described hereininclude the step of administering to a subject or patient in need ofsuch treatment an effective amount of a compound described by theformulae herein. Antagonism of various vasopressin receptor subtypes hasbeen associated with numerous physiological and therapeutic benefits.These benefits may arise from antagonism of both peripheral and centralnervous system vasopressin receptors. Peripheral nervous systemutilities include administration of vasopressin V_(1a) and/orvasopressin V₂ antagonists as adjuncts in heart failure or asantithrombotic agents. Central nervous system effects includeadministration of vasopressin V_(1a) and/or vasopressin V_(1b)antagonists of the compounds described herein for the treatment ofobsessive-compulsive disorder, aggressive disorders, depression,anxiety, and other psychological and neurological disorders.

Illustrative disease states that are responsive to the antagonism of avasopressin V₂ receptor and treatable by the methods described hereininclude various cardiovascular diseases, including, disorders orconditions associated with platelet aggregation, and the like. Inaddition, methods for treating other disease states and conditionstreatable by for example oxytocin receptor antagonism, tachykininreceptor antagonism, neurokinin 1 receptor antagonism, neurokinin 2receptor antagonism, and the like are described herein, where the methodincludes the step of administering to a patient in need of relief fromsuch a disease state or condition an effective amount of one or moresubstituted 2-(azetidin-2-on-1-yl)alkanedioic acids, substituted2-(azetidin-2-on-1-yl)hydroxyalkylalkanoic acids, substituted2-(azetidin-2-on-1-yl)alkylalkanoic acids, and analogs and derivativesthereof described herein.

Method Example 1

Human vasopression VIb receptor-expressing cells. Human vasopressinreceptor 1B (HV1B) cDNA (see, Lolait et al., “Extrapituitary expressionof the rat V1b vasopressin receptor gene” Proc. Natl. Acad. Sci. USA.92:6783-7 (1995); de Keyzer et al., “Cloning and characterization of thehuman V3(V1b) pituitary vasopressin receptor” FEBS Lett. 356:215-20(1994); Sugimoto et al., “Molecular cloning and functional expression ofa cDNA encoding the human V1b vasopressin receptor” J. Biol. Chem.269:27088-92 (1994)) was inserted into a mammalian cell expressionvector PCI-neo (Promega) at EcoR1 site. The recombinant plasmid carryingHV1B cDNA was identified from transformed E. Coli clones and used forthe transfection of Chinese hamster ovary cell (CHO-K1, ATCC). Twomicrograms of HV1B receptor DNA was introduced into 10⁵ CHO cellscultured in 6-well plate, using Fugene-6 mediated transfection technique(Boehringer Mannheim). Twenty-four hrs post transfection, Cells werethen cultured under selection of G-418 (0.25 mg/ml) supplemented to theculture medium. Three days later, limited dilution was carried out toobtain single cell clones in 96-well plates. After a period of 2-weeksof growth, monoclones were expanded into two sets of 12-well plates.When confluence was reached, one set of wells were assayed for theirability to bind tritium-labeled arginine-vasopressin (NEN). Ninepositive clones were initially identified out of 60 clones screened, andclones that demonstrated highest AVP binding were saved as permanentcell lines for HV1B affinity screening of Serenix compounds.

Method Example 2

Human or rat vasopression V_(1a), V_(1b), and/or V₂ cell-based receptorbinding assay. The V_(1a), V_(1b), and/or V₂ cell lines (cellsexpressing either the human or rat V_(1a), V_(1b), and/or V₂ receptors)were grown in alpha-MEM medium supplemented with 10% fetal bovine serumand 250 ug/ml G418 (Gibco, Grand Island, N.Y.) in 75 cm² flask. Forcompetitive binding assay, hV1b cells were dissociated with enzyme-free,PBS based cell dissociation solution (Specialty Media, Phillipursburg,N.J.), following the manufacturer's protocol. Cells were plated into12-well culture plates at a rate of one flask to 18 plates (rate shouldbe adjusted according to the extent of confluency), and maintained inculture for 2-3 days. Culture medium was then removed, cells were washedonce with 2 ml binding buffer (25 mM Hepes, 0.25% BSA, 1×DMEM, PH=7.0)at room temperature. To each well, 990 ul binding buffer containing 1 nM³H-AVP was added, and followed by the addition of 10 ul series dilutedtesting compounds or cold AVP, all dissolved in DMSO. All incubationswere in triplicate, and dose-inhibition curves consisted of totalbinding (DMSO only) and 5 concentrations (0.1, 1.0, 10, 100, and 1000nm) of test agent, or cold AVP, encompassing the IC50. Cells wereincubated for 30 min at 37° C. in a moisturized incubator. Assay mixturewas then removed and each well was washed three times with PBS (pH=7.4).After washing, 1 ml 2% SDS was added per well and plates were let sitfor 15 min at RT. Gently pat the plate to make sure that lysed cellswere detached. The whole content in a well was transferred to ascintillation vial. Each well was then rinsed with 0.5 ml PBS and addedto the corresponding vial. Scintillation fluid (Ecoscint, NationalDiagnostics, Atlanta, Ga.) was then added at 3 ml per vial. Samples werecounted in a liquid scintillation counter (Beckman LS3801). IC50 and Kivalues were calculated using Prism Curve fitting software.

Selected examples were tested in these assay on cells expressing humanV_(1a) or human V_(1b) receptors. Binding affinities (IC₅₀) forillustrative compounds are summarized in the following Table. Inhibitionconstants (K_(i)) for illustrative compounds are also summarized in thefollowing Table.

Human V_(1a) Human V_(1a) Human V_(1b) Human V_(1b) Binding BindingBinding Binding Affinity Affinity Affinity Affinity Example (IC₅₀ (nM))(K_(i) (nM)) (IC₅₀ (μM)) (K_(i) (μM)) 9B 0.11 0.07 1.10 0.69

Method Example 3

Inhibition of vasopressin V_(1b)-mediated phosphatidylinositol turnover,a functional assay for antagonist activity. The physiological effects ofvasopressin are mediated through specific G-protein coupled receptors.The vasopressin V_(1a), V_(1b), and/or V₂ receptors are coupled to a Gprotein, which is coupled to cAMP. The agonist or antagonist characterof the compounds described herein may be determined by their ability toinhibit vasopressin-mediated turnover of phosphatidylinositol by usingconventional methods, including the procedure described in the followingparagraphs.

Cells expressing the human or rat V_(1a), V_(1b), and/or V₂ receptorsare grown in alpha-modified minimal essential medium containing 10%fetal bovine serum and 0.25 mg/ml G418. Three days prior to the assay,near-confluent cultures are dissociated and seeded in 6-well tissueculture plates, about 100 wells being seeded from each 75 cm² flask(equivalent to 12:1 split ratio). Each well contains 1 ml of growthmedium with 2 μCi of [³H]myo-inositol (American Radiolabeled Chemicals,St. Louis, Mo.).

All assays are in triplicate except for basal and 10 nM AVP (both n=6).Arginine vasopressin (AVP) is dissolved in 0.1N acetic acid. Candidatedrugs are dissolved in DMSO on the day of the experiment and diluted inDMSO to 200 times the final test concentration. Candidate drugs and AVP(or corresponding volumes of DMSO) are added separately as 5 ul in DMSOto 12×75 mm glass tubes containing 1 ml of assay buffer (Tyrode'sbalanced salt solution containing 50 mM glucose, 10 mM LiCl, 15 mM HEPESpH 7.4, 10 uM phosphoramidon, and 100 uM bacitracin). The order ofincubations are randomized. Incubations are initiated by removing theprelabeling medium, washing the monolayer once with 1 ml of 0.9% NaCl,and adding the contents of the assay tubes. The plates are incubated for1 hr at 37° C. Incubations are terminated by removing the incubationmedium and adding 500 ul of ice cold 5% (w/v) trichloroacetic acid andallowing them to stand for 15 min.

The incubates are fractionated on BioRad Poly-Prep Econo-Columns packedwith 0.3 ml of AG 1X-8100-200 formate resin. Resin is mixed 1:1 withwater and 0.6 ml added to each column. Columns are then washed with 10ml water. Scintillation vials (20 ml) are placed under each column. Foreach incubation well, the contents are transferred to a minicolumn,after which the well is washed with 0.5 ml distilled water, which isalso added to the minicolumn. The columns are then washed twice with 5ml of 5 mM myo-inositol to elute free inositol. A 1 ml aliquot of thisis transferred to a new 20 ml scintillation vial, plus 10 ml of BeckmanReady Protein Plus, and counted. After the myo-inositol wash iscomplete, empty scintillation vials are placed under the columns, and[³H] inositol phosphates are eluted with three additions of 1 ml 0.5 Mammonium formate containing 0.1 N formic acid. Elution conditions areoptimized to recover inositol mono-, bis-, and trisphosphates, withouteluting the more metabolically inert tetrakis-, pentakis-, andhexakis-phosphates. Samples are counted in a Beckman LS 6500multipurpose scintillation counter after addition of 10 ml Tru-CountHigh Salt Capacity scintillation fluid.

Inositol lipids are measured by adding 1 ml of 2% sodium dodecyl sulfate(SDS) to each well, allowing the wells to sit for at least 30 min. Lysedcontent in each well is transferred to a 20 ml scintillation vial. 10 mlBeckman Ready Protein Plus scintillation fluid is added andradioactivity counted.

Concentration-response curves for AVP and concentration-inhibitioncurves for test agents versus 10 nM AVP were analyzed by nonlinearleast-squares curve-fitting to a 4-parameter logistic function.Parameters for basal and maximal inositol phosphates, EC₅₀ or IC₅₀, andHill coefficient were varied to achieve the best fit. The curve-fittingwas weighted under the assumption that the standard deviation wasproportional to dpm of radioactivity. Full concentration-response curvesfor AVP were run in each experiment, and IC₅₀ values were converted toK_(i) values by application of the Cheng-Prusoff equation, based on theEC₅₀ for AVP in the same experiment. Inositol phosphates were expressedas dpm per 10⁶ dpm of total inositol incorporation.

Experiments to test for competitively of test agents consisted ofconcentration-response curves for AVP in the absence and presence of twoor more concentrations of test agent. Data were fit to the followingcompetitive logistic equation:

$Y = {B + \frac{M \times \left\{ {A/\left\lbrack {E + \left( {D/K} \right)} \right\rbrack} \right\}^{Q}}{1 + \left\{ {A/\left\lbrack {E + \left( {D/K} \right)} \right\rbrack} \right\}^{Q}}}$where Y is dpm of inositol phosphates, B is concentration of basalinositol phosphates, M is the maximal increase in concentration ofinositol phosphates, A is the concentration of agonist (AVP), E is theEC₅₀ for agonist, D is the concentration of the antagonist, K is theK_(i) for antagonist, and Q is the cooperativity (Hill coefficient).

Experiments to test for competition by test agents consist ofconcentration-response curves for AVP in the absence and presence of atleast five concentrations of test agent. Ki values, which reflect theantagonistic activities against AVP in the production of signalingmolecule IP3, are calculated with prism software based on Cheng andPrusoff equation.

Method Example 4

Seed finding by golden hamsters. It is appreciated that a hamster'sability to find seeds under certain conditions may reflect their levelof anxiety. This method for assaying seed finding capabilities inhamsters treated with the compounds described herein is an animal modelof anxiety.

Male, Syrian golden hamsters (Mesocricetus auratus) (120-130 g) obtainedfrom Harlan Sprague-Dawley Laboratories (Indianapolis, Ind.) are housedindividually in Plexiglas cages (24 cm×24 cm×20 cm), maintained on areverse light:dark (cycle (14:10; lights on at 19:00 hr), and providedfood and water ad libitum. All tests are conducted during the dark phaseof the circadian cycle under dim red illumination. Prior to testing, allanimals are fasted for 20-24 hrs. Ninety min after intraperitoneal (IP)injection of SRX262 (n=10) or saline vehicle (n=10), animals are takenfrom their home cage and placed into a holding cage for 2 min, Duringtheir absence, six sunflower seeds were buried under the bedding in onecorner of their home cage. Animals are placed back into their home cagerandomly facing any one of the empty corners and timed for their latencyto find the seeds during a five minute observation period. The latencyto find the seeds is reduced following treatment with the compoundsdescribed herein and comparable in magnitude to fluoxetine, buspirone,and chlordiazapoxide.

Method Example 5

Social Subjugation in Hamsters, a biochemical marker assay. There is abody of literature on the neuroendocrine and behavioral consequences ofrepeated social subjugation in adult male golden hamsters. In adultanimals, losing fights and being relegated to low social status is verystressful, resulting in altered levels of adrenal and gonadal steroidstogether with changes in social behaviors (Rose et al., 1975; Eberhartet al., 1980, 1983). Studies on adult male hamsters show depressedlevels of testosterone and elevated levels of glucocorticoids followingrepeated defeat by dominant conspecifics (Huhman et al., 1991).

Male hamsters are housed and maintained as described above. For 30minutes each day for fourteen consecutive days, animals are exposed tothreat and attack from a larger conspecific (n=14). Following thesedaily episodes of traumatic stress animals are left undisturbed in theirhome cages for ten days. During this recovery period animals are treatedwith the compounds described herein (1 mg/kg/day) (n=7) or salinevehicle (n=7). At the end of this treatment period animals aresacrificed by decapitation and trunk blood collected for theradioimmunoassay of testosterone and cortisol. The testosterone levelsof chronically subjugated hamsters are very low while the basal cortisollevels are high. This neuroendocrine profile is altered by treatmentwith the compounds described herein. The collected data indicate thatblocking V_(1b) receptors can enhance recovery from traumatic stresslike social subjugation.

Method Example 6

Social Subjugation in Hamsters, a behavioral assay, screening forantidepression-like activity. The hamster model of social subjugation inthe resident intruder paradigm is used. The resident/intruder model ofaggression relies on the motivation of a resident animal to chase andfight intruders coming into their territory (Miczek 1974). Smalleranimals placed into the home cage of a resident will be more easilydefeated and become socially subjugated with repeated encounters. Socialsubjugation is a significant and natural stressor in the animal kingdom.Animals defeated and subjugated during establishment of dominancehierarchies or territorial encounters can be highly submissive in futureagonistic interactions.

For example, defeated mice display less aggression and more submissivebehavior (Frishlnecht et al., 1982; Williams and Lierle 1988). Ratsconsistently defeated by more aggressive conspecifics show a behavioralinhibition characterized by less social initiative and offensiveaggression, as well as an increase in defensive behavior (Van de Poll etal., 1982). Repeatedly defeated male hamsters respond in a submissivemanner when confronted by a non-aggressive intruder (Potegal et al.,1993), in addition their normal reproductive behavior is reduced asmeasured by latency to mount a receptive female. Moreover, followingrepeated defeat by a dominant conspecific, a resident hamster will bedefensive or fearful of smaller-sized non aggressive intruders (Potegalet al., 1993). The generalization of submissive behavior towardnon-threatening, novel stimulus animals is an example of “conditioneddefeat” (Potegal et al., 1993). Conditioned defeat in adult hamsters isnot permanent as the flight and defensive behaviors disappear over manyweeks. Animals displaying conditioned defeat are treated with thecompounds described herein, and observed for a return to normalaggressive and reproductive behaviors.

In addition, social subjugation has a pronounced effect on the animal'sneuroendocrinology. In adult animals, losing fights and being relegatedto low social status alters levels of adrenal and gonadal steroids (Roseet al., 1975; Eberhart et al., 1908, 1983). Adult male hamsters showdepressed levels of testosterone and elevated levels of glucocorticoidsfollowing repeated defeat by dominant conspecifics (Huhman et al.,1991). Recovery of normal testosterone and cortisol levels is assessedin animals treated with the compounds described herein.

Male, Syrian golden hamsters (Mesocricetus auratus) (120-130 g) obtainedfrom Harlan Sprague-Dawley Laboratories (Indianapolis, Ind.) are housedindividually in Plexiglas cages (24 cm×24 cm×20 cm), maintained on areverse light:dark cycle (14:10; lights on at 19:00 hr), and providedfood and water ad libitum. All tests are conducted during the dark phaseof the circadian cycle under dim red illumination. Each compound istested in 3 doses (100 μg, 1 mg, and 10 mg/kg) plus saline vehicle.Twenty-four animals (six per group) are tested. Animals are sociallysubjugated by placing them into the home cage of a larger hamster eachday for 30 min for 14 consecutive days. Animals are exposed to adifferent resident each day so that the threat and attack isunremitting. Following the cessation of social subjugation, animals areallowed to recover undisturbed in their home cage for the next twoweeks. During this time they are treated with a compound describedherein, or with vehicle for one week. At the end of the week, theanimals are tested for aggression toward a smaller intruder placed intotheir home cage. Animals are scored for latency to bite, number ofbites, and contract time. On the following day, a receptive female isplaced into the animal's home cage, and the animal is scored for latencyto mount. At the end of two weeks, animals are sacrificed and trunkblood assayed for testosterone and cortisol. All animals are sacrificedduring the first 2 hrs of the dark phase of the light:dark cycle tominimize circadian variations in cortisol levels. The data betweentreatments is compared with one-way, ANOVA followed by Bonferroni posthoc tests.

Method Example 7

Elevated Plus Maze. The elevated plus-maze was developed for screeninganxiolytic and anxiogenic drug effects in rodents. The method has beenvalidated behaviorally, physiologically, and pharmacologically. Theplus-maze consists of two open arms and two enclosed arms. Rats and micetend to naturally make fewer entries into the open arms than into theclosed arms and will spend significantly less time in open arms.Confinement to the open arms is associated with significantly moreanxiety-related behavior and higher stress hormone levels thanconfinement to the closed arms. Clinically effective anxiolytics, e.g.,chlordiazepoxide or diazepam, significantly increase the percentage oftime spent in the open arms and the number of entries into the openarms. Conversely, anxiogenic compounds like yohimbin or amphetaminesreduce open arm entries and time spent in the open arms.

Male mice are group housed in a normal 12:12 light:dark cycle with lighton at 0800 hr and provided food and water ad libitum. The plus-mazeconsists of two open arms, 40 cm long, 6 cm wide with no walls. The twoclosed arms have the same dimensions with walls 25 cm high. Each pair ofarms are arranged opposite to each other to form the plus-maze. The mazeis elevated to a height of 50 cm. Each drug is tested in 3 doses (100μg, 1 mg, and 10 mg/kg) plus saline vehicle. Twenty-four animals (sixper group) are tested in the plus-maze 90 min following the IP injectionin a volume of ca. 0.1 ml. At the start of the experiment, the animal isplaced at the end of one of the open arms. Over a five min observationperiod, animals are scored for the latency to enter the closed arm, timespent in the closed arm, and the number of open arm entries followingthe first occupation of the closed arm. The data between treatments arecompared with one-way, ANOVA followed by Bonferroni post hoc tests.

Method Example 8

Impulsivity/Inappropriate Aggression. Impulsivity and/or inappropriateaggression may be determined using standard animal behavior assays,including the resident-intruder paradigm, the isolation inducedaggression paradigm, and the interfemale aggression and/or intermaleaggression paradigms. These assays may be applied to mice, rats, and/orhamsters. Arginine vasopressin (AVP) has been implicated in theaggressive behaviors of a number of species, including humans (see,Coccaro et al., “Cerebrospinal fluid vasopressin levels: correlates withaggression and serotonin function in personality-disordered subjects”Arch. Gen. Psychiatry 55:708-14 (1998)). Infusions of AVP receptorsantagonists have been shown to reduce aggression (see, Ferris &Potengal, “Vasopressin receptor blockade in the anterior hypothalamussuppresses aggression in hamsters” Physiol. Behav. 44:235-39 (1988)). Astudy of the vasopressin V_(1b) knockout mouse indicated reductions inaggressive behavior by these animals (see, Wersinger et al.,“Vasopressin V_(1b) receptor knockout reduces aggressive behavior inmale mice” Mol. Psychiatry. 7:975-84 (2002)).

Adult male Syrian hamsters (Mesocricetus auratus, Charles RiverLaboratories) are used as subjects. Hamsters to be used as residents arehoused individually for at least 2 weeks prior to the beginning of theexperiment. A subpopulation of smaller males are used as intruders,which are group-housed (three/cage) in order to minimize aggressionlevels. Resident and intruder pairs should have a minimum of about a10-g weight difference. For example, the weight range for residents isbetween 105 and 150 g, and intruder weights ranged from 95 to 140 g,although these absolute weights may vary. Animals are housed inPlexiglas cages (46.0×24.0×21.0 cm) with corn cob bedding in atemperature (e.g. 69° F.) and humidity-controlled room with food andwater available ad libitum, which is maintained on a 14:10 light-darkcycle with lights off at 12:00 noon. Tests are run under red lightillumination during the first 3 h of the dark phase of the light-darkcycle. All animals are handled daily for 10 days prior to the start ofthe study.

A single nondrug screening test (resident-intruder) is run with eachindividually housed hamster to determine the baseline levels ofaggression of the animal. Only resident males that show a minimum of onebite during the test session are used in the drug test. Tests with thecompounds described herein are run 48 h after the screening test.Twenty-five minutes after drug administration, residents are moved tothe testing room. Intruders are introduced into the resident home cage 5min later, for a 10-min test. Each resident is confronted with adifferent intruder than was used in the screening phase. It is to beunderstood that the protocols used in this experiment are in compliancewith the applicable state and federal regulations. Behavioral measuresinclude attack latency, latency to bite, and number of bites. Data areanalyzed by one-way ANOVA, optionally followed by Newman-Keuls post hoctests. Further details of this assay are found in Blanchard et al., “AVPV1b selective antagonist SSR149415 blocks aggressive behaviors inhamsters” Pharmacol., Biochem. Behav. 80:189-94 (2005).

Method Example 9

Human oxytocin binding and functional assay. Oxytocin is known for itshormonal role in parturition and lactation. Oxytocin agonists are usefulclinically to induce lactation; induce or augment labor; controlpostpartum uterine atony and hemorrhage; cause uterine contraction aftercesarean section or during other uterine surgery; and to inducetherapeutic abortion. Oxytocin, acting as a neurotransmitter in thecentral nervous system, also plays an important role in the expressionof central functions such as maternal behavior, sexual behavior(including penile erection, lordosis and copulatory behavior), yawning,tolerance and dependence mechanisms, feeding, grooming, cardiovascularregulation and thermoregulation (Argiolas and Gessa, Neuroscience andBiobehavioral Reviews, 15:217-231 (1991)). Oxytocin antagonists findtherapeutic utility as agents for the delay or prevention of prematurelabor; or to slow or arrest delivery for brief periods in order toundertake other therapeutic measures.

Compounds described herein are also believed to be oxytocin agents.Oxytocin preparations and a number of oxytocin agonists are commerciallyavailable for therapeutic use. In recent years, oxytocin antagonistswith antiuterotonic activity have been developed and evaluated for theirpotential use in the treatment of preterm labor and dysmenorrhyea (Pavoet al., J. Med. Chem., 37:255-259 (1994); Akerlund et al., Br. J.Obstet. Gynaecol., 94:1040-1044 (1987); Akerlund et al., Br. J. Obstet.Gynaecol., 86:484-487 (1979)). The oxytocin antagonist atosiban has beenstudied clinically and resulted in a more significant inhibition ofpreterm contractions than did placebo (Goodwin et al., Am. J. Obstet.Gynecol., 170:474 (1994)).

The human oxytocin receptor has been cloned and expressed (Kimura etal., Nature, 356:526-529 (1992)), it is identified under the accessionnumber X64878. To demonstrate the affinity of the compounds describedherein for the human oxytocin receptor, binding studies were performedusing a cell line expressing the human oxytocin receptor in 293 cells(henceforth referred to as the OTR cell line) substantially by theprocedure described by Morel et al. (Nature, 356:523-526 (1992)). The293 cell line is a permanent line of primary human embryonal kidneycells transformed by sheared human adenovirus type 5 DNA. It isidentified as ATCC CRL-1533.

The OTR cell line was grown in DMEM (Delbecco's Modified EssentialMedium, Sigma, St. Louis, Mo., USA) with 10% fetal bovine serum, 2 mML-glutamine, 200 μg hygromycin (Sigma, St. Louis, Mo., USA) and 250μg/ml G418 (Gibco, Grand Island, N.Y., USA). To prepare membranes, OTRcells were grown to confluency in 20 roller bottles. Cells weredissociated with enzyme-free cell dissociation medium (Specialty Media,Lavallette, N.J., USA) and centrifuged at 3200 rpm for 15 minutes. Thepellet was resuspended in 40 mL of Tris-HCl(tris[hydroxymethyl]aminomethane hydrochloride) buffer (50 mM, pH 7.4)and homogenized for 1 minute with a Tekmar Tissumizer (Cincinnatti, OhioUSA). The suspension was centrifuged at 40,000×g for 10 minutes. Thepellet was resuspended and centrifuged as above. The final pellet wassuspended in 80 mL of Tris 7.4 buffer and stored in 4 mL aliquots at−80° C. For assay, aliquots were resuspended in assay buffer and dilutedto 375 μg protein per mL. Protein concentration was determined by BCAassay (Pierce, Rockford, Ill., USA).

Assay buffer was 50 mM Tris-HCl (tris[hydroxymethyl]aminomethanehydrochloride), 5 mM MgCl₂, and 0.1% bovine serum albumin at pH 7.4. Theradioligand for binding assays was [³H]oxytocin([tyrosyl-2,6-³H]oxytocin, 48.5 Ci/mmol, DuPont NEN, Boston, Mass.,USA). The order of additions was 195 μL assay buffer, 200 μL OTRmembranes (75 μg protein) in assay buffer, 5 μL of test agent indimethylsulfoxide (DMSO) or DMSO alone, and 100 μL [³H]oxytocin in assaybuffer (final concentration 1.0 nM). Incubations were for one hour atroom temperature. Bound radioligand was separated from free byfiltration on a Brandel cell harvester (Gaithersburg, Md., USA) throughWhatman GF/B glass-fiber filters that had been soaked for 2 hours in0.3% polyethylenimine. The filters were washed with ice-cold 50 mMTris-HCl (pH 7.7 at 25° C.) and the filter circles were placed inscintillation vials, to which were then added 5 mL Ready Protein Plus™scintillation fluid, and counted in a liquid scintillation counter. Allincubations were in triplicate, and dose-inhibition curves consisted oftotal binding, nonspecific binding (100 μM oxytocin, Sigma, St. Louis,Mo., USA), and 6 or 7 concentrations of test agent encompassing theIC₅₀. Total binding was typically about 1,000 cpm and nonspecificbinding about 200 cpm. IC₅₀ values were calculated by nonlinearleast-squares curve-fitting to a 4-parameter logistic model. Certaincompounds of formula (I) have shown affinity for the oxytocin receptor.

Several bioassays are available to determine the agonist or antagonistcharacter of compounds exhibiting affinity at the oxytocin receptor. Onesuch assay is described in U.S. Pat. No. 5,373,089, hereby incorporatedby reference. Said bioassay is derived from procedures described in apaper by Sawyer et al. (Endocrinology, 106:81 (1980)), which in turn wasbased on a report of Holton (Brit. J. Pharmacol., 3:328 (1948)). Theassay calculations for pA₂ estimates are described by Schild (Brit. J.Pharmacol, 2:189 (1947)).

Method Example 10 Assay for Oxytocin Functional Activity

1. Animals: a 1.5 cm piece of uterus from a virgin rat (Holtzman) innatural estrus is used for the assay.

2. Buffer/Assay Bath: The buffer used is Munsicks. This buffer contains0.5 mM Mg²⁺. The buffer is gassed continuously with 95% oxygen/5% carbondioxide giving a pH of 7.4. The temperature of the assay bath is 37° C.A 10 mL assay bath is used that contains a water jacket for maintainingthe temperature and inlet and outlet spikets for adding and removingbuffer.

3. Polygraph/transducer: The piece of uterine tissue used for the assayis anchored at one end and connected to a Statham Strain Gauge ForceTransducer at the other end which in turn is attached to a GrassPolygraph Model 79 for monitoring the contractions.

4. Assay Protocol:

(a) The tissue is equilibrated in the assay bath for one hour withwashing with new buffer every 15 minutes. One gram of tension is kept onthe tissue at all times.

(b) The tissue is stimulated initially with oxytocin at 10 nM toacclimate the tissue and with 4 mM potassium chloride (KCl) to determinethe maximum contractile response.

(c) A cumulative dose response curve is then done with oxytocin and aconcentration of oxytocin equivalent to approximately 80% of the maximumis used for estimating the pA₂ of the antagonist.

(d) The tissue is exposed to oxytocin (Calbiochemical, San Diego,Calif.) for one minute and washed out. There is a three minute intervalbefore addition of the next dose of agonist or antagonist. When theantagonist is tested, it is given five minutes before the agonist. Theagonist is given for one minute. All responses are integrated using a7P10 Grass Integrator. A single concentration of oxytocin, equal to 80%of the maximum response, is used to test the antagonist. Three differentconcentrations of antagonists are used, two that will reduce theresponse to the agonist by less than 50% and one that will reduce theresponse greater than 50% (ideally this relation would be 25%, 50% and75%). This is repeated three times for each dose of antagonist for athree point assay.

(e) Calculations for pA₂− The dose-response (DR) ratios are calculatedfor antagonist and a Schild's Plot is performed by plotting the Log(DR-1) vs. Log of antagonist concentration. The line plotted iscalculated by least-squares regression analysis. The pA₂ is theconcentration of antagonist at the point where the regression linecrosses the 0 point of the Log (DR-1) ordinate. The pA₂ is the negativeLog of the concentration of antagonist that will reduce the response tothe agonist by one-half.

Method Example 11

Tachykinin Receptor Binding Assay Compounds described herein arebelieved to be tachykinin agents. Tachykinins are a family of peptideswhich share a common amidated carboxy terminal sequence. Substance P wasthe first peptide of this family to be isolated, although itspurification and the determination of its primary sequence did not occuruntil the early 1970's. Between 1983 and 1984 several groups reportedthe isolation of two novel mammalian tachykinins, now termed neurokininA (also known as substance K, neuromedin 1, and neurokinin α), andneurokinin B (also known as neuromedin K and neurokinin β). See, J. E.Maggio, Peptides, 6 (Supplement 3): 237-243 (1985) for a review of thesediscoveries.

Tachykinin receptor antagonists are of value in the treatment of a widevariety of clinical conditions which are characterized by the presenceof an excess of tachykinin. These clinical conditions may includedisorders of the central nervous system such as anxiety, depression,psychosis, and schizophrenia; neurodegenerative disorders such asdementia, including senile dementia of the Alzheimer's type, Alzheimer'sdisease, AIDS-associated dementia, and Down's syndrome; demyelinatingdiseases such as multiple sclerosis and amyotrophic lateral sclerosisand other neuropathological disorders such as peripheral neuropathy,such as diabetic and chemotherapy-induced neuropathy, and post-herpeticand other neuralgias; acute and chronic obstructive airway diseases suchas adult respiratory distress syndrome, bronchopneumonia, bronchospasm,chronic bronchitis, drivercough, and asthma; inflammatory diseases suchas inflammatory bowel disease, psoriasis, fibrositis, osteoarthritis,and rheumatoid arthritis; disorders of the musculo-skeletal system, suchas osteoporosis; allergies such as eczema and rhinitis; hypersensitivitydisorders such as poison ivy; ophthalmic diseases such asconjunctivitis, vernal conjunctivitis, and the like; cutaneous diseasessuch as contact dermatitis, atopic dermatitis, urticaria, and othereczematoid dermatites; addiction disorders such as alcoholism;stress-related somatic disorders; reflex sympathetic dystrophy such asshoulder/hand syndrome; dysthymic disorders; adverse immunologicalreactions such as rejection of transplanted tissues and disordersrelated to immune enhancement or suppression such as systemic lupuserythematosis; gastrointestinal disorders or diseases associated withthe neuronal control of viscera such as ulcerative colitis, Crohn'sdisease, emesis, and irritable bowel syndrome; disorders of bladderfunction such as bladder detrusor hyper-reflexia and incontinence;artherosclerosis; fibrosing and collagen diseases such as sclerodermaand eosinophilic fascioliasis; irritative symptoms of benign prostatichypertrophy; disorders of blood flow caused by vasodilation andvasospastic diseases such as angina, migraine, and Raynaud's disease;and pain or nociception, for example, that attributable to or associatedwith any of the foregoing conditions, especially the transmission ofpain in migraine.

Tachykinins are widely distributed in both the central and peripheralnervous systems. When released from nerves, they exert a variety ofbiological actions, which, in most cases, depend upon activation ofspecific receptors expressed on the membrane of target cells.Tachykinins are also produced by a number of non-neural tissues. Themammalian tachykinins substance P, neurokinin A, and neurokinin B actthrough three major receptor subtypes, denoted as NK-1, NK-2, and NK-3,respectively. These receptors are present in a variety of organs.

Substance P is believed inter alia to be involved in theneurotransmission of pain sensations, including the pain associated withmigraine headaches and with arthritis. These peptides have also beenimplicated in gastrointestinal disorders and diseases of thegastrointestinal tract such as inflammatory bowel disease. Tachykininshave also been implicated as playing a role in numerous other maladies,as discussed infra.

In view of the wide number of clinical maladies associated with anexcess of tachykinins, the development of tachykinin receptorantagonists will serve to control these clinical conditions. Theearliest tachykinin receptor antagonists were peptide derivatives. Theseantagonists proved to be of limited pharmaceutical utility because oftheir metabolic instability. Recent publications have described novelclasses of non-peptidyl tachykinin receptor antagonists which generallyhave greater oral bioavailability and metabolic stability than theearlier classes of tachykinin receptor antagonists. Examples of suchnewer non-peptidyl tachykinin receptor antagonists are found in EuropeanPatent Publication 591,040 A1, published Apr. 6, 1994; PatentCooperation Treaty publication WO 94/01402, published Jan. 20, 1994;Patent Cooperation Treaty publication WO 94/04494, published Mar. 3,1994; Patent Cooperation Treaty publication WO 93/011609, published Jan.21, 1993, Patent Cooperation Treaty publication WO 94/26735, publishedNov. 24, 1994. Assays useful for evaluating tachykinin receptorantagonists are well known in the art. See, e.g., J. Jukic et al., LifeSciences, 49:1463-1469 (1991); N. Kucharczyk et al., Journal ofMedicinal Chemistry, 36:1654-1661 (1993); N. Rouissi et al., Biochemicaland Biophysical Research Communications, 176:894-901 (1991).

Method Example 12

NK-1 Receptor Binding Assay. NK-1 antagonists are useful in thetreatment of pain, especially chronic pain, such as neuropathic pain,post-operative pain, and migraines, pain associated with arthritis,cancer-associated pain, chronic lower back pain, cluster headaches,herpes neuralgia, phantom limb pain, central pain, dental pain,neuropathic pain, opioid-resistant pain, visceral pain, surgical pain,bone injury pain, pain during labor and delivery, pain resulting fromburns, including sunburn, post partum pain, angina pain, andgenitourinary tract-related pain including cystitis.

In addition to pain, NK-1 antagonists are especially useful in thetreatment and prevention of urinary incontinence; irritative symptoms ofbenign prostatic hypertrophy; motility disorders of the gastrointestinaltract, such as irritable bowel syndrome; acute and chronic obstructiveairway diseases, such as bronchospasm, bronchopneumonia, asthma, andadult respiratory distress syndrome; artherosclerosis; inflammatoryconditions, such as inflammatory bowel disease, ulcerative colitis,Crohn's disease, rheumatoid arthritis, osteoarthritis, neurogenicinflammation, allergies, rhinitis, cough, dermatitis, urticaria,psoriasis, conjunctivitis, emesis, irritation-induced miosis; tissuetransplant rejection; plasma extravasation resulting from cytokinechemotherapy and the like; spinal cord trauma; stroke; cerebral stroke(ischemia); Alzheimer's disease; Parkinson's disease; multiplesclerosis; amyotrophic lateral sclerosis; schizophrenia; anxiety; anddepression.

Radioreceptor binding assays were performed using a derivative of apreviously published protocol. D. G. Payan et al, Journal of Immunology,133:3260-3265 (1984). In this assay an aliquot of IM9 cells (1×10⁶cells/tube in RPMI 1604 medium supplemented with 10% fetal calf serum)was incubated with 20 pM ¹²⁵I-labeled substance P in the presence ofincreasing competitor concentrations for 45 minutes at 4° C.

The IM9 cell line is a well-characterized cell line which is readilyavailable to the public. See, e.g., Annals of the New York Academy ofScience, 190:221-234 (1972); Nature (London), 251:443-444 (1974);Proceedings of the National Academy of Sciences (USA), 71:84-88 (1974).These cells were routinely cultured in RPMI 1640 supplemented with 50μg/mL gentamicin sulfate and 10% fetal calf serum.

The reaction was terminated by filtration through a glass fiber filterharvesting system using filters previously soaked for 20 minutes in 0.1%polyethylenimine. Specific binding of labeled substance P was determinedin the presence of 20 nM unlabeled ligand.

Method Example 13

NK-2 Receptor Binding Assay. NK-2 antagonists are useful in thetreatment of urinary incontinence, bronchospasm, asthma, adultrespiratory distress syndrome, motility disorders of thegastrointestinal tract, such as irritable bowel syndrome, and pain.

The CHO-hNK-2R cells, a CHO-derived cell line transformed with the humanNK-2 receptor, expressing about 400,000 such receptors per cell, weregrown in 75 cm² flasks or roller bottles in minimal essential medium(alpha modification) with 10% fetal bovine serum. The gene sequence ofthe human NK-2 receptor is given in N. P. Gerard et al., Journal ofBiological Chemistry, 265:20455-20462 (1990).

For preparation of membranes, 30 confluent roller bottle cultures weredissociated by washing each roller bottle with 10 ml of Dulbecco'sphosphate buffered saline (PBS) without calcium and magnesium, followedby addition of 10 ml of enzyme-free cell dissociation solution(PBS-based, from Specialty Media, Inc.). After an additional 15 minutes,the dissociated cells were pooled and centrifuged at 1,000 RPM for 10minutes in a clinical centrifuge. Membranes were prepared byhomogenization of the cell pellets in 300 mL 50 mM Tris buffer, pH 7.4with a TEKMAR® homogenizer for 10-15 seconds, followed by centrifugationat 12,000 RPM (20,000×g) for 30 minutes using a BECKMAN JA-14® rotor.The pellets were washed once using the above procedure. and the finalpellets were resuspended in 100-120 mL 50 mM Tris buffer, pH 7.4, and 4ml aliquots stored frozen at −70° C. The protein concentration of thispreparation was 2 mg/mL.

For the receptor binding assay, one 4-mL aliquot of the CHO-hNK-2Rmembrane preparation was suspended in 40 mL of assay buffer containing50 mM Tris, pH 7.4, 3 nM manganese chloride, 0.02% bovine serum albumin(BSA) and 4 μg/mL chymostatin. A 200 μL volume of the homogenate (40 μgprotein) was used per sample. The radioactive ligand was[¹²⁵I]iodohistidyl-neurokinin A (New England Nuclear, NEX-252), 2200Ci/mmol. The ligand was prepared in assay buffer at 20 nCi per 100 μL;the final concentration in the assay was 20 pM. Non-specific binding wasdetermined using 1 μM eledoisin. Ten concentrations of eledoisin from0.1 to 1000 nM were used for a standard concentration-response curve.

All samples and standards were added to the incubation in 10 μLdimethylsulfoxide (DMSO) for screening (single dose) or in 5 μL DMSO forIC₅₀ determinations. The order of additions for incubation was 190 or195 μL assay buffer, 200 μL homogenate, 10 or 5 μL sample in DMSO, 100μL radioactive ligand. The samples were incubated 1 hr at roomtemperature and then filtered on a cell harvester through filters whichhad been presoaked for two hours in 50 mM Tris buffer, pH 7.7,containing 0.5% BSA. The filter was washed 3 times with approximately 3mL of cold 50 mM Tris buffer, pH 7.7. The filter circles were thenpunched into 12×75 mm polystyrene tubes and counted in a gamma counter.

Method Example 14

Treatment of Emesis. In addition to the above indications the compoundsdescribed herein may be useful in the treatment of emesis, includingacute, delayed, or anticipatory emesis, such as emesis induced bychemotherapy, radiation, toxins, pregnancy, vestibular disorders,motion, surgery, migraine, and variations in intercranial pressure. Inparticular, the compounds of the formulae described herein may be of usein the treatment of emesis induced by antineoplastic (cytotoxic) agentsincluding those routinely used in cancer chemotherapy.

Examples of such chemotherapeutic agents include alkylating agents, forexample, nitrogen mustards, ethyleneimine compounds, alkyl sulfonates,and other compounds with an alkylating action, such as nitrosoureas,cisplatin, and dacarbazine; antimetabolites, for example, folic acid,purine, or pyrimidine antagonists; mitotic inhibitors, for example vincaalkaloids and derivatives of podophyllotoxin; and cytotoxic antibiotics.

Particular examples of chemotherapeutic agents are described, forinstance, by D. J. Stewart in NAUSEA AND VOMITING: RECENT RESEARCH ANDCLINICAL ADVANCES, (J. Kucharczyk et al., eds., 1991), at pages 177-203.Commonly used chemotherapeutic agents include cisplatin, dacarbazine(DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin,daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, andchlorambucil. R. J. Gralla et al., Cancer Treatment Reports, 68:163-172(1984).

The compounds of the formulae described herein may also be of use in thetreatment of emesis induced by radiation, including radiation therapysuch as in the treatment of cancer, or radiation sickness; and in thetreatment of post-operative nausea and vomiting.

Method Example 15

Inhibition of platelet aggregation. Vasopressin V₂ receptors are alsoknown to mediate platelet aggregation. Vasopressin receptor agonistscause platelet aggregation, while vasopressin V₂ receptor antagonistsinhibit the platelet aggregation precipitated by vasopressin orvasopressin agonists. The degree of antagonist activity of the compoundsdescribed herein may be determined by using conventional methods,including the assay described in the following paragraphs.

Blood from healthy, human volunteers was collected by venipuncture andmixed with heparin (60 mL of blood added to 0.4 mL of heparinized salinesolution (4 mg heparin/mL saline)). Platelet-rich plasma (PRP) wasprepared by centrifuging whole blood (150×g), and indomethacin (3 μM)was added to PRP to block the thromboxane-mediated release reaction. PRPwas continuously stirred at 37° C. and change in optical density wasfollowed after the addition of arginine vasopressin (AVP) (30 nM) toinitiate aggregation. Compounds were dissolved in 50% dimethylsulfoxide(DMSO) and added (10 μL/415 μL PRP) before the addition of AVP. Thepercent inhibition of AVP-induced aggregation was measured and an IC₅₀calculated.

In studies using washed platelets, 50 mL of whole blood was mixed with10 mL of citrate/heparin solution (85 mM sodium citrate, 64 mM citricacid, 111 mM glucose, 5 units/mL heparin) and PRP isolated as describedabove. PRP was then centrifuged (150×g) and the pellet resuspended in aphysiologic buffer solution (10 mM HEPES, 135 mM sodium chloride, 5 mMpotassium chloride, and 1 mM magnesium chloride) containing 10 μMindomethicin. Human fibrinogen (0.2 mg/mL) and calcium chloride (1 mM)were added to stirred platelets before initiating aggregation with AVP(30 nM) as previously described.

Method Example 16

Flank marking behavior in golden hamsters. Obsessive-compulsive diseaseappears in a great variety of degrees and symptoms, generally linked bythe victim's uncontrollable urge to perform needless, ritualistic acts.Acts of acquiring, ordering, cleansing and the like, beyond any rationalneed or rationale, are the outward characteristic of the disease. Abadly afflicted subject may be unable to do anything but carry out therituals required by the disease. Obsessive-compulsive disease, in allits variations, is a preferred target of treatment with the presentadjunctive therapy method and compositions. The utility of the compoundsof Formula (I) in the treatment of obsessive-compulsive disorder wasdemonstrated as described in the following assay.

In golden hamsters, a particular stereotypy, flank marling behavior, canbe induced by microinjections of vasopressin (10-100 mL, 1-100 μM) intothe anterior hypothalamus (Ferris et al., Science, 224, 521-523 (1984);Albers and Ferris, Regulatory Peptides, 12, 257-260 (1985); Ferris etal., European Journal of Pharmacology, 154, 153-159 (1988)). Followingthe releasing stimulus, the behavior is initiated by grooming, lickingand combing of the large sebaceous glands on the dorsolateral flanks.Bouts of flank gland grooming may be so intense that the flank region isleft matted and soaked in saliva. After grooming, the hamsters displayflank marking behavior, a type of scent marking involved in olfactorycommunication (Johnston, Physio. Behav., 51, 437-448 (1985); Ferris etal., Physio. Behav., 40, 661-664 (1987)), by arching the back andrubbing the flank glands vigorously against any vertical surface.Vasopressin-induced flank marking is usually induced within a minuteafter the microinjection (Ferris et al., Science, 224, 521-523 (1984)).The behavior is specific to vasopressin, as micro-injections of otherneuropeptides, excitatory amino acids, and catecholamines do not elicitflank marking (Ferris et al., Science, 224, 521-523 (1984); Albers andFerris, Regulatory Peptides, 12, 257-260 (1985)). Furthermore, flankmarking is specific to the vasopressin V₁ receptor, as the behavior isselectively inhibited by V₁ receptor antagonists and activated by V₁receptor agonists (Ferris et al., Neuroscience Letters, 55, 239-243(1985); Albers et al., Journal of Neuroscience, 6, 2085-2089 (1986);Ferris et al., European Journal of Pharmacology, 154, 153-159 (1988)).

All animals in this assay are adult male golden hamsters (Mesocricetusauratus) weighing approximately 160 gm. The animals undergo stereotaxicsurgery, and are allowed to recover before behavioral testing. Thehamsters are kept on a reverse light cycle (14 hr light, 10 hr dark,lights on at 19:00) in Plexiglas™ cages, and receive food and water adlibitum.

Stereotaxic surgery is performed under pentobarbital anesthesia. Thestereotaxic coordinates are: 1.1 mm anterior to the bregma, 1.8 mmlateral to the midsagittal suture at an 8° angle from the vertical line,and 4.5 mm below the dura. The nose bar is placed at the level of theinteraural line. An unilateral 26-gauge guide cannula is lowered to thesite and secured to the skull with dental cement. The guide cannulae areclosed with a 33-gauge obturator extending 1 mm beyond the guide. Theinnercanulae used for the microinjections extends 3.0 mm beyond theguide to reach the anterior hypothalamus.

The hamsters are microinjected with 1 μM vasopressin in a volume of 150mL. The vasopressin is given as a cocktail with 200 mM, 20 mM, 2 mM ofthe test compound or alone, in the vehicle, dimethylsulfoxide. Both thevasopressin and the test compound are dissolved in 100%dimethylsulfoxide. All injections are aimed at the anteriorhypothalamus. Animals are scored for flank marking for a period of 10minutes in a clean cage.

Method Example 17

Use in combination with a serotonin reuptake inhibitor. Another aspectof this invention is the use of compounds of Formula (I) in combinationwith a serotonin reuptake inhibitor for use in the treatment ofobsessive-compulsive disease, aggressive disorder, or depression.Compounds useful as serotonin reuptake inhibitors include but are notlimited to:

Fluoxetine, N-methyl-3-(p-trifluoromethylphenoxy)-3-phenylpropylamine,is marketed in the hydrochloride salt form, and as the racemic mixtureof its two enantiomers. U.S. Pat. No. 4,314,081 is an early reference onthe compound. Robertson et al., J. Med. Chem., 31, 1412 (1988), taughtthe separation of the R and S enantiomers of fluoxetine and showed thattheir activity as serotonin uptake inhibitors is similar to each other.In this document, the word “fluoxetine” will be used to mean any acidaddition salt or the free base, and to include either the racemicmixture or either of the R and S enantiomers;

Duloxetine, N-methyl-3-(1-naphthalenyloxy)-3-(2-thienyl)propanamine, isusually administered as the hydrochloride salt and as the (+)enantiomer. It was first taught by U.S. Pat. No. 4,956,388, which showsits high potency. The word “duloxetine” will be used here to refer toany acid addition salt or the free base of the molecule;

Venlafaxine is known in the literature, and its method of synthesis andits activity as an inhibitor of serotonin and norepinephrine uptake aretaught by U.S. Pat. No. 4,761,501. Venlafaxine is identified as compoundA in that patent;

Milnacipran (N,N-diethyl-2-aminomethyl-1-phenylcyclopropanecarboxamide)is taught by U.S. Pat. No. 4,478,836, which prepared milnacipran as itsExample 4. The patent describes its compounds as antidepressants. Moretet al., Neuropharmacology, 24, 1211-19 (1985), describe itspharmacological activities as an inhibitor of serotonin andnorepinephrine reuptake;

Citalopram,1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-5-isobenzofurancarbonitrile,is disclosed in U.S. Pat. No. 4,136,193 as a serotonin reuptakeinhibitor. Its pharmacology was disclosed by Christensen et al., Eur. J.Pharmacol., 41, 153 (1977), and reports of its clinical effectiveness indepression may be found in Dufour et al., Int. Clin. Psychopharmacol.,2, 225 (1987), and Timmerman et al., ibid., 239;

Fluvoxamine, 5-methoxy-1-[4-(trifluoromethyl)phenyl]-1-pentanoneO-(2-aminoethyl)oxime, is taught by U.S. Pat. No. 4,085,225. Scientificarticles about the drug have been published by Claassen et al., Brit. J.Pharmacol., 60, 505 (1977); and De Wilde et al., J. Affective Disord.,4, 249 (1982); and Benfield et al., Drugs, 32, 313 (1986);

Paroxetine,trans-(−)-3-[(1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine,may be found in U.S. Pat. Nos. 3,912,743 and 4,007,196. Reports of thedrug's activity are in Lassen, Eur. J. Pharmacol., 47, 351 (1978);Hassan et al., Brit. J. Clin. Pharmacol., 19, 705 (1985); Laursen etal., Acta Psychiat. Scand., 71, 249 (1985); and Battegay et al.,Neuropsychobiology, 13, 31 (1985); and

Sertraline,(1S-cis)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-N-methyl-1-naphthylaminehydrochloride, a serotonin reuptake inhibitor disclosed in U.S. Pat. No.4,536,518, is marketed as an antidepressant.

All of the above-referenced patents are hereby incorporated byreference.

The adjunctive therapy of this aspect of the present invention iscarried out by administering a vasopressin V_(1a) antagonist describedherein together with a serotonin reuptake inhibitor in any manner thatprovides effective levels of the compounds in the body at the same time.All of the compounds concerned are orally available and are normallyadministered orally, and so oral administration of the adjunctivecombination is preferred. They may be administered together, in a singledosage form, or may be administered separately.

This aspect of the present invention provides a potentiation of thedecrease in the concentration of vasopressin observed as an effect ofadministration of a vasopressin V_(1a) antagonist by administration of aserotonin reuptake inhibitor. This aspect of the present invention isparticularly suited for use in the treatment of depression and obsessivecompulsive disorder. Such disorders may often be resistant to treatmentwith a serotonin reuptake inhibitor alone.

While it is possible to administer a compound employed in the methodsdescribed herein directly without any formulation, the compounds areusually administered in the form of pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and at least oneactive ingredient. These compositions can be administered by a varietyof routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Many of the compoundsemployed in the methods described herein are effective as bothinjectable and oral compositions. Such compositions are prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, (16thed. 1980).

In making the pharmaceutical compositions used in the methods describedherein, the active ingredient is usually mixed with an excipient,diluted by an excipient, or enclosed within such a carrier which can bein the form of a capsule, sachet, paper, or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacinth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Thecompositions described herein can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 0.05 to about 100 mg, more usually about1.0 to about 30 mg, of the active ingredient. The term “unit dosageform” refers to physically discrete units suitable as unitary dosagesfor human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

The active compounds are generally effective over a wide dosage range.For example, dosages per day normally fall within the range from about0.01 to about 30 mg/kg of body weight. In illustrative variations,dosages per day may fall in the range from about 0.02 to about 10 mg/kgof body weight, in the range from about 0.02 to about 1 mg/kg of bodyweight, or in the range from about 0.02 to about 0.1 mg/kg of bodyweight. Such dosage ranges are applicable for the treatment of anypatient or mammal. In addition, for the treatment of adult humans,illustrative doses fall in the range from about 0.02 to about 15 mg/kgof body weight, or in the range from about 0.1 to about 10 mg/kg/day, insingle or divided dose. However, it is to be understood that the amountof the compound actually administered will be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compound orcompounds administered, the age, weight, and response of the individualpatient, and the severity of the patient's symptoms, and therefore theabove dosage ranges are intended to be illustrative are not intended toand should not be interpreted to limit the invention in any way. In someinstances dosage levels below the lower limit of the aforesaid range maybe more than adequate, while in other cases still larger doses may beemployed without causing any harmful side effect. It is appreciated thatsuch larger doses may be first divided into several smaller doses foradministration throughout the day.

The type of formulation employed for the administration of the compoundsemployed in the methods described herein may be dictated by theparticular compounds employed, the type of pharmacokinetic profiledesired from the route of administration and the compound(s), and thestate of the patient.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Compound of formula (I) 30.0 Starch305.0 Magnesium stearate 5.0The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Compound of formula (I) 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Compound of formula (I) 5 Lactose 95The active mixture is mixed with the lactose and the mixture is added toa dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Compound of formula (I) 30.0 mg  Starch45.0 mg  Microcrystalline cellulose 35.0 mg  Polyvinylpyrrolidone (as10% solution in water) 4.0 mg Sodium carboxymethyl starch 4.5 mgMagnesium stearate 0.5 mg Talc 1.0 mg Total 120 mg The active ingredient, starch, and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50-60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Compound of formula (I)  40.0 mg Starch109.0 mg Magnesium stearate  1.0 mg Total 150.0 mgThe active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Quantity Ingredient (mg) Compound of formula (I)   25 mg Saturated fattyacid glycerides to 2,000 mgThe active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose aremade as follows:

Quantity Ingredient (mg) Compound of formula (I) 50.0 mg Xanthan gum 4.0mg Sodium carboxymethyl cellulose(11%) 50.0 mg Microcrystallinecellulose (89%) Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Colorq.v. Purified water to 5.0 mlThe medicament, sucrose, and xanthan gum are blended, passed through aNo. 10 mesh U.S. sieve, and then mixed with a previously made solutionof the microcrystalline cellulose and sodium carboxymethyl cellulose inwater. The sodium benzoate, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation Example 8

Capsules, each containing 15 mg of medicament, are made as follows:

Quantity Ingredient (mg/capsule) Compound of formula (I)  15.0 mg Starch407.0 mg Magnesium stearate  3.0 mg Total 425.0 mgThe active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 425 mg quantities.

Formulation Example 9

An intravenous formulation may be prepared as follows:

Quantity Ingredient (mg) Compound of formula (I) 250.0 mg Isotonicsaline 1000 ml

Formulation Example 10

A topical formulation may be prepared as follows:

Quantity Ingredient (mg) Compound of formula (I) 1-10 g Emulsifying Wax30 g Liquid Paraffin 20 g White Soft Paraffin to 100 gThe white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Formulation Example 11

Sublingual or buccal tablets, each containing 10 mg of activeingredient, may be prepared as follows:

Quantity Ingredient (mg/tablet) Compound of formula (I)  10.0 mgGlycerol 210.5 mg Water 143.0 mg Sodium Citrate  4.5 mg PolyvinylAlcohol  26.5 mg Polyvinylpyrrolidone  15.5 mg Total 410.0 mgThe glycerol, water, sodium citrate, polyvinyl alcohol, andpolyvinylpyrrolidone are admixed together by continuous stirring andmaintaining the temperature at about 90° C. When the polymers have goneinto solution, the resulting solution is cooled to about 50-55° C. andthe medicament is slowly admixed. The homogenous mixture is poured intoforms made of an inert material to produce a drug-containing diffusionmatrix having a thickness of about 2-4 mm. This diffusion matrix is thencut to form individual tablets having the appropriate size.

Formulation Example 12

In the methods described herein, another illustrative formulationemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofthe compounds described herein in controlled amounts. The constructionand use of transdermal patches for the delivery of pharmaceutical agentsis well known in the art. See, e.g., U.S. Pat. No. 5,023,252, issuedJun. 11, 1991, herein incorporated by reference. Such patches may beconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Formulation Example 13

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system, used for the transport ofbiological factors to specific anatomical regions of the body, isdescribed in U.S. Pat. No. 5,011,472, which is herein incorporated byreference.

Formulation Example 14

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs or prodrugs.Latentiation is generally achieved through blocking of the hydroxy,carbonyl, sulfate, and primary amine groups present on the drug torender the drug more lipid soluble and amenable to transportation acrossthe blood-brain barrier. Alternatively, the delivery of hydrophilicdrugs may be enhanced by intra-arterial infusion of hypertonic solutionsthat can transiently open the blood-brain barrier.

While the invention has been illustrated and described in detail in theforegoing description, such an illustration and description is to beconsidered as illustrative and exemplary and not restrictive incharacter, it being understood that only the illustrative embodimentshave been shown and described and that all changes and modificationsthat come within the spirit of the invention are desired to beprotected.

1. A compound of the formula

wherein: Q is oxygen, sulfur, —S(O)— or —SO₂—; n is 1 or 2; A ismonosubstituted amino, disubstituted amino, or an optionally substitutednitrogen-containing heterocycle attached at a nitrogen; R¹ is hydrogenor C₁-C₆ alkyl; R² is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, halo, haloalkyl, cyano, formyl, alkylcarbonyl,alkoxycarbonyl, or a substituent selected from the group consisting of—CO₂R⁸, —CONR⁸R^(8′), and —NR⁸(COR⁹); R³ is selected from the groupconsisting of

wherein R¹⁰ and R¹¹ are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, alkoxyalkyl, alkylcarbonyloxy, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted arylalkyloxy, optionally substituted arylalkylcarbonyloxy,diphenylmethoxy, and triphenylmethoxy; and R¹² is selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, alkoxycarbonyl,optionally substituted aryloxycarbonyl, optionally substitutedarylalkyl, and optionally substituted aryloyl; R⁴ is alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, alkylcarbonyl, optionally substitutedaryl, optionally substituted arylalkyl, optionally substitutedarylhaloalkyl, optionally substituted arylalkoxyalkyl, optionallysubstituted arylalkenyl, optionally substituted arylhaloalkenyl, oroptionally substituted arylalkynyl; R^(5′) is selected from the groupconsisting of —SR¹⁵, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, (C₁-C₄alkoxy)-(C₁-C₄ alkyl), optionally-substituted arylalkyl, heterocyclyl,heterocyclyl(C₁-C₄ alkyl), and R^(6′)R^(7′)N—(C₂-C₄ alkyl); whereheterocyclyl is in each occurrence independently selected from the groupconsisting of tetrahydrofuryl, morpholinyl, pyrrolidinyl, piperidinyl,piperazinyl, homopiperazinyl, or quinuclidinyl; where said morpholinyl,pyrrolidinyl, piperidinyl, piperazinyl, or homopiperazinyl is optionallyN-substituted with C₁-C₄ alkyl or optionally substituted aryl(C₁-C₄alkyl); R^(6′) is hydrogen or alkyl, and R^(7′) is alkyl, cycloalkyl,optionally substituted aryl, or optionally substituted arylalkyl; orR^(6′) and R^(7′) are taken together with the attached nitrogen atom toform an heterocycle selected from the group consisting of pyrrolidinyl,piperidinyl, morpholinyl, piperazinyl, and homopiperazinyl; where saidpiperazinyl or homopiperazinyl is optionally N-substituted with R^(13′);R⁸ and R^(8′) are each independently selected in each instance fromhydrogen, alkyl, cycloalkyl, optionally substituted aryl, or optionallysubstituted arylalkyl; or R⁸ and R^(8′) are taken together with theattached nitrogen atom to form an heterocycle selected from the groupconsisting of optionally substituted pyrrolidinyl, piperidinyl,morpholinyl, piperazinyl, and homopiperazinyl; R⁹ is selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, alkoxyalkyl, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl, andR⁸R^(8′)N—(C₁-C₄ alkyl); R^(13′) is selected from the group consistingof hydrogen, alkyl, cycloalkyl, alkoxycarbonyl, optionally substitutedaryloxycarbonyl, optionally substituted arylalkyl, and optionallysubstituted aryloyl; R¹⁵ is selected from the group consisting of C₁-C₆alkyl, C₃-C₈ cycloalkyl, (C₁-C₄ alkoxy)-(C₁-C₄ alkyl),optionally-substituted aryl(C₁-C₄ alkyl), heterocyclyl,heterocyclyl(C₁-C₄ alkyl), and R^(6′)R^(7′)N—(C₂-C₄ alkyl); whereheterocyclyl is in each occurrence independently selected from the groupconsisting of tetrahydrofuryl, morpholinyl, pyrrolidinyl, piperidinyl,piperazinyl, homopiperazinyl, or quinuclidinyl; where said morpholinyl,pyrrolidinyl, piperidinyl, piperazinyl, or homopiperazinyl is optionallyN-substituted with C₁-C₄ alkyl or optionally substituted aryl(C₁-C₄alkyl); and hydrates, solvates, and pharmaceutically acceptable saltsthereof; provided that when Q is oxygen, then n is 2 and R^(5′) is not—SR¹⁵.
 2. A compound of the formula

wherein: Aryl is an optionally substituted monocyclic or polycyclicaromatic group; m is 1, 2, or 3; A is monosubstituted amino,disubstituted amino, or an optionally substituted nitrogen-containingheterocycle attached at a nitrogen; R¹ is hydrogen or C₁-C₆ alkyl; R² ishydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, halo, haloalkyl,cyano, formyl, alkylcarbonyl, alkoxycarbonyl, or a substituent selectedfrom the group consisting of —CO₂R⁸, —CONR⁸R^(8′), and —NR⁸(COR⁹); R³ isselected from the group consisting of

wherein R¹⁰ and R¹¹ are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, alkoxyalkyl, alkylcarbonyloxy, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted arylalkyloxy, optionally substituted arylalkylcarbonyloxy,diphenylmethoxy, and triphenylmethoxy; and R¹² is selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, alkoxycarbonyl,optionally substituted aryloxycarbonyl, optionally substitutedarylalkyl, and optionally substituted aryloyl; R⁴ is alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, alkylcarbonyl, optionally substitutedaryl, optionally substituted arylalkyl, optionally substitutedarylhaloalkyl, optionally substituted arylalkoxyalkyl, optionallysubstituted arylalkenyl, optionally substituted arylhaloalkenyl, oroptionally substituted arylalkynyl; R⁸ and R^(8′) are each independentlyselected in each instance from hydrogen, alkyl, cycloalkyl, optionallysubstituted aryl, or optionally substituted arylalkyl; or R⁸ and R^(8′)are taken together with the attached nitrogen atom to form anheterocycle selected from the group consisting of optionally substitutedpyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, andhomopiperazinyl; R⁹ is selected from the group consisting of hydrogen,alkyl, cycloalkyl, alkoxyalkyl, optionally substituted aryl, optionallysubstituted arylalkyl, optionally substituted heteroaryl, optionallysubstituted heteroarylalkyl, and R⁸R^(8′)N—(C₁-C₄ alkyl); andpharmaceutically acceptable salts thereof.
 3. The compound of claim 1wherein Q is oxygen.
 4. The compound of claim 1 wherein Q is sulfur andn is
 1. 5. The compound of claim 1 wherein Q is sulfur and n is
 2. 6.The compound of claim 1 wherein R^(5′) is C₁-C₄ alkyl.
 7. The compoundof claim 1 wherein R^(5′) is optionally substituted arylalkyl.
 8. Thecompound of claim 1 wherein R^(5′) is an optionally substitutedaryl(C₁-C₂)alkyl.
 9. The compound of claim 1 wherein R^(5′) is benzyl.10. The compound of claim 1 wherein R^(5′) is an optionally substitutedheteroaryl(C₁-C₂ alkyl).
 11. The compound of claim 1 wherein A is anoptionally substituted nitrogen-containing heterocycle.
 12. The compoundof claim 1 wherein A is a substituted heterocycle, wherein theheterocycle is selected from the group consisting of pyrrolidinyl,piperidinyl, and piperazinyl.
 13. The compound of claim 1 wherein A ispiperidinyl or piperazinyl, and where A is substituted in the 4-positionwith pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl,pyrrolidinyl(C₁-C₄ alkyl), piperidinyl(C₁-C₄ alkyl), piperazinyl(C₁-C₄alkyl), or homopiperazinyl(C₁-C₄ alkyl).
 14. The compound of claim 1wherein R³ is a structure selected from the group consisting of


15. The compound of claim 14 wherein R³ is


16. The compound of claim 1 wherein R⁴ is optionally substitutedaryl(C₁-C₄ alkyl), optionally substituted aryl(C₂-C₄ alkenyl), oroptionally substituted aryl(C₂-C₄ alkynyl).
 17. The compound of claim 16wherein R⁴ is optionally substituted phenyl(C₂-C₄ alkenyl).
 18. Thecompound of claim 2 wherein Aryl is optionally substituted phenyl. 19.The compound of claim 2 wherein Aryl is optionally substituted thienyl.20. The compound of claim 2 wherein m is
 1. 21. The compound of claim 2wherein m is
 3. 22. A pharmaceutical composition comprising the compoundof claim 1 and a pharmaceutically acceptable carrier, diluent,excipient, or a combination thereof.